Pengisian poin C sampai dengan poin H mengikuti template berikut dan tidak dibatasi jumlah kata atau halaman namun disarankan seringkas mungkin. Dilarang menghapus/memodifikasi template ataupun menghapus penjelasan di setiap poin.
Ekstraksi RNA Total dan RT-PCR Fragmen Gen Penyandi Aktin
RNA total dari R. trisperma diisolasi dengan menggunakan Total RNA Mini Kit (Plant Geneaid) dan dilanjutkan dengan pendekatan RT-PCR. Proses RT-PCR didahului dengan reverse transcriptase terhadap molekul mRNA sehingga diperoleh molekul complementary DNA (cDNA). Molekul cDNA tersebut kemudian digunakan sebagai cetakan dalam proses PCR [1]. Hasil ekstraksi RNA total pada endoperma biji R. trisperma menghasilkan konsentrasi sebesar 173,4 ng/µL dengan perbandingan A260/A280 sebesar 2,00. Tahap sintesis utas pertama cDNA dilakukan dengan menggunakan 0,69 µg RNA total dengan kondisi reaksi seperti pada tabel 3.2. Selanjutnya, dilakukan amplifikasi PCR dengan cDNA sebagai template. Kondisi reaksi proses PCR ditunjukkan pada tabel 3.6 dan tidak dilakukan optimasi suhu annealing (Ta) pada proses amplifikasi. Primer yang digunakan untuk proses amplifikasi cDNA gen penyandi aktin adalah primer degenerate dikarenakan tidak tersedianya data informasi genetik aktin R. trisperma di database GenBank [2]. Primer degenerate didesain dengan metode penjajaran beberapa sekuen (multiple sequence alignment) gen yang sama pada spesies-spesies yang berkerabat dekat [3]. Penjajaran sekuen gen penyandi aktin dilakukan terhadap empat spesies anggota famili Euphorbiaceae, yakni Ricinus communis, Hevea brasiliensis, Jatropha curcas, dan Vernicia fordii. Penjajaran sekuen dilakukan untuk mendapatkan daerah konservatif dengan nilai degeneracy yang rendah [4-5]. Daerah konservatif hasil penjajaran digunakan sebagai dasar pemilihan primer dikarenakan lebih spesifik [6]. Sekuen gen aktin yang telah disejajarkan dan telah dilakukan pemotongan nukleotida yang tidak rata adalah sebesar 1069 bp. Kandidat primer yang diperoleh dari daerah konservatif adalah sebagai berikut :
Tabel 4.4 Kandidat primer gen penyandi aktin R. trisperma
*Kode genap: Primer forward; kode ganjil: primer reverse
Kandidat primer pada tabel 4.4 telah memenuhi parameter desain primer, yakni memiliki ukuran panjang antara 18-28 basa, rentang Tm 50-650C, serta kadar GC berkisar 40-60% [7-9]. Pasangan kandidat primer forward (10ACTks) dan primer reverse (15ACTks) menghasilkan amplikon dengan C. HASIL PELAKSANAAN PENELITIAN: Tuliskan secara ringkas hasil pelaksanaan penelitian yang telah
dicapai sesuai tahun pelaksanaan penelitian. Penyajian meliputi data, hasil analisis, dan capaian luaran (wajib dan atau tambahan). Seluruh hasil atau capaian yang dilaporkan harus berkaitan dengan tahapan pelaksanaan penelitian sebagaimana direncanakan pada proposal. Penyajian data dapat berupa gambar, tabel, grafik, dan sejenisnya, serta analisis didukung dengan sumber pustaka primer yang relevan dan terkini.
Kode Sekuen Primer Posisi Ukura Tm GC n (0C) (%) 5’-GCTGGGTTT 10ACTks GCTGGTGATGAT 61-82 22 bp 59 54,5 G-3’ 5’-GCAAGGATA 1025- 15ACTks GATCCTCCAATC 22 bp 54,9 50 1046 C-3’
panjang 986 bp kemudian digunakan untuk mengamplifikasi gen penyandi aktin R.trisperma melalui proses RT-PCR. Hasil amplifikasi kemudian dielektroforesis menggunakan gel agarose 2% untuk memvisualisasikan hasil PCR. Elektroforegram produk RT-PCR sampel R. trisperma direpresentasikan pada gambar 4.4
Gambar 4.4 Elektroforegram produk RT-PCR fragmen gen penyandi aktin R. trsiperma. Keterangan: M: Marka; A: Fragmen gen penyandi aktin R. trsiperma
Visualisasi hasil RT-PCR menggunakan pasangan primer 10ACTks dan 15ACTks menghasilkan pita tunggal dengan panjang fragmen sesuai prediksi. Fragmen gen yang berhasil diisolasi adalah 986 bp sesuai hasil kombinasi primer 10ACTks dan 15ACTks. Pita yang terbentuk dari proses RT-PCR tersebut diduga merupakan pita gen target. Namun, untuk lebih yakin bahwa pita berukuran 986 bp tersebut merupakan gen penyandi aktin R. trisperma perlu dilakukan pengurutan sekuen nukleotida dan dianalisis dengan bioinformatika [10-12]. Sampel produk RT-PCR selanjutnya dikirim ke perusahaan Toyobo Bio Technology untuk dilakukan kloning dan sekuensing.
Kloning Fragmen Gen Penyandi Aktin R. trisperma ke dalam Plasmid pTA2
Tahapan pengkloningan meliputi elektroforesis produk PCR dengan gel agarose, purifikasi produk PCR, ligasi, transformasi, seleksi transforman, PCR koloni, isolasi plasmid, dan sekuensing. Elektroforesis produk PCR dilakukan dengan menggunakan gel agarose 1% dalam buffer TBE 1X. Hasil visualisasi memperlihatkan fragmen DNA yang berukuran 986 bp (Gambar 4.5).
Gambar 4.5 Hasil elektroforesis produk PCR fragmen gen penyandi aktin R. trisperma Keterangan: M: Marka 10 kb; 1: fragmen gen penyandi aktin R. trisperma
Produk PCR selanjutnya tersebut dipurifikasi dengan menggunakan Zymo DNA Clean and Concentrator (Zymo Research). Tujuan purifikasi adalah untuk membersihkan sisa-sisa kelebihan primer, dNTPs, enzim, amplikon DNA pendek karena polimerisasi yang tidak sempurna, pewarna (dye), dan sisa garam pada reaksi PCR [13]. Hasil purifikasi selanjutnya digunakan sebagai DNA insert pada proses ligasi. Ligase dilakukan dengan cara menggabungkan fragmen DNA dengan vektor pengkloningan pTA2 (Toyobo). Vektor plasmid ini disebut juga vektor T karena memiliki basa timin (T) pada ujung 3’ sehingga vektor ini dapat menempel dengan fragmen DNA hasil amplifikasi yang memiliki kelebihan basa adenin (A) tanpa adanya tahap pemotongan dengan enzim restriksi [14]. Proses ligase dikatalisis oleh enzim ligase sehingga terbentuk plasmid rekombinan. Vektor pTA2 memiliki ukuran 2981 bp dan DNA insert aktin berukuran 986 bp, sehingga menghasilkan vektor rekombinan berukuran 3967 bp. Hasil ligasi tidak divisualisasikan dengan elektroforesis gel agarose sebab langsung digunakan untuk proses transformasi.
Proses transformasi dilakukan dengan menggunakan E. coli strain DH5α yang telah dibuat kompeten dengan menggunakan kit Mix and Go Competent CellsTM (Zymo Research). Plasmid DNA rekombinan ditambahkan sedikit demi sedikit ke dalam suspensi E. coli kompeten sambil dihomogenkan, kemudian di kultur pada medium LB padat yang telah ditambahkan antibiotik ampisillin. Tahap selanjutnya yaitu seleksi transforman dengan metode koloni putih biru (blue white screening) pada media yang mengandung antibiotik ampisilin, IPTG, dan X-gal. Keberhasilan penyisipan fragmen DNA ditunjukkan oleh adanya koloni putih yang tumbuh pada media seleksi [15]. Koloni yang terbentuk pada media seleksi adalah bakteri yang memiliki plasmid rekombinan. Bakteri tersebut berhasil ditransformasi, sedangkan yang tidak tertransformasi akan mati akibat perlakuan ampisilin [7]. Seleksi biru putih terjadi ketika dua fragmen yang inaktif bersatu membentuk enzim β-galaktosidase yang fungsional. Enzim tersebut menghidrolisis laktosa menjadi glukosa. Aktifitas enzim tersebut dapat diuji dengan menggunakan senyawa 5-bromo-4-kloro-3-indolil- β-D-galaktosidase (X-gal) yang menghasilkan warna biru pada
medium. Enzim tersebut dihasilkan oleh gen lacZ pada MCS vektor pengkloningan. Isopropil-1-tio- β-galaktosidase (IPTG) digunakan sebagai induser untuk menonaktifkan repressor lacZ. Bakteri yang mengandung plasmid rekombinan tidak menghasilkan enzim β-galaktosidase sehingga pada medium akan berwarna putih, sedangkan bakteri yang tidak mengandung plasmid rekombinan tetap menghasilkan enzim β-galaktosidase yang dapat memecah senyawa X-gal, sehingga koloni akan berwarna biru [7].
Seleksi transforman dengan metode blue white screening belum tentu sesuai dengan teori [16]. Hal ini dikarenakan produk yang dihasilkan pada proses ligasi tidak saja mengandung plasmid rekombinan tetapi juga produk-produk lain, seperti vektor yang tidak tersambung (unligated vektor), fragmen DNA yang tidak tersambung dengan vektor (tidak terligasi), vektor yang tersambung tetapi tidak mengandung gen sisipan (self ligated), dan plasmid rekombinan dengan gen sisipan yang berbeda [17]. Oleh karena itu, perlu dilakukan proses PCR koloni untuk menghindari kesalahan seleksi transforman. Tahap ini sebagai konfirmasi untuk meyakinkan bahwa fragmen DNA telah berhasil disisipkan [16]. Melalui proses ini, gen yang disisipkan diamplifikasi menggunakan primer spesifik dengan terlebih dahulu dilakukan lisis sel [18]. Proses PCR koloni hampir sama dengan PCR biasa, hanya saja pada pada proses ini perlu dilakukan lisis sel rekombinan terlebih dahulu dengan mesin PCR. Kit yang digunakan untuk proses PCR koloni adalah KOD FX Neo (Toyobo). Sebanyak 8 koloni putih dipilih untuk di PCR, adapun primer yang digunakan pada proses PCR ini adalah primer T3 promotor dan T7 promotor. Hasil PCR kemudian divisualisasi dengan gel agarose 1% dalam buffer TBE 1X. Gambar 4.3 memperlihatkan bahwa fragmen DNA telah berhasil disisipkan ke E. coli DH5α pada koloni ke 1, 5, 6, 7, dan 8. Koloni 6 dipilih untuk dilanjutkan ke proses isolasi plasmid dan sekuensing dikarenakan koloni 6 memperlihatkan elektroforegram pita DNA yang berukuran sesuai target, yaitu 1173 bp.
Gambar 4.6 Hasil elektroforesis PCR koloni plasmid rekombinan Keterangan: M: Marka 10 kb; 1-8: Produk PCR koloni
Ukuran 1173 bp tersebut menunjukkan terdapat penambahan sebesar 187 bp dari DNA insert yang hanya berukuran 986 bp. Hal tersebut disebabkan verifikasi insert DNA aktin dengan PCR menggunakan primer yang berasal dari vektor yaitu T3 promotor dan T7 promotor. Gambar 4.7 menunjukkan skema pelekatan primer T3 promotor dan T7 promotor yang menyebabkan terjadi penambahan sebesar 187 bp.
Gambar 4.7 Skema pelekatan primer T3 promotor dan T7 promotor terhadap DNA insert
Produk PCR pada koloni 6 dilakukan isolasi plasmid dengan menggunakan kit ZR Plasmid MiniPrep (Zymo Research). Pada dasarnya isolasi DNA plasmid prinsipnya hampir sama dengan isolasi DNA kromosom. Hanya saja terdapat perbedaan dalam segi ukuran antara DNA plasmid dengan DNA kromosom. Perbedaan yang paling signifikan adalah dari segi ukuran. Ukuran DNA plasmid jauh lebih kecil dibandingkan dengan DNA kromosom. Selain itu, DNA plasmid memiliki perbedaan konformasi dengan DNA kromosom [19]. Prinsip utama isolasi plasmid adalah mengeluarkan plasmid DNA dari dalam sel bakteri dengan cara melisiskan sel bakteri tersebut tanpa membuat DNA plasmid rusak [20].
Plasmid yang telah berhasil diisolasi kemudian dilakukan proses sekuensing. Sekuensing plasmid dilakukan dengan menggunakan metode dideoksi Sanger. Adapun primer spesifik yang digunakan pada proses sekuensing adalah primer T7 promotor dan T3 promotor. Data hasil sekuensing berupa kromatogram yang menggambarkan urutan basa nukleotida fragmen gen penyandi aktin R. trisperma. Hasil pengurutan sekuen frgmen DNA yang diperoleh menunjukkan bahwa ukuran gen yang teramplifikasi adalah 986 bp. Lampiran 3 menampilkan sekuen gen penyandi aktin tanaman R. trisperma yang berhasil diisolasi. Warna hijau merupakan sekuen insert gen penyandi aktin R. trisperma, sedangkan warna kuning menunjukkan sekuen primer. Analisis penyejajaran (alignment) sekuen DNA aktin R. trsiperma dengan sekuen DNA lengkap dari famili Euphorbiaceae; Jatropha curcas, Ricinus communis, dan Hevea brasiliensis, masih terdapat selisih sebesar 148 bp. Berdasarkan hasil tersebut, diprediksi masih terdapat fragmen gen penyandi aktin R. trisperma yang belum berhasil diisolasi. Isolasi cDNA lengkap fragmen gen penyandi aktin pada tanaman telah dilakukan dengan menggunakan metode RACE (Rapid Amplification of cDNA Ends) (Cazorla et al., 2015).
D. STATUS LUARAN: Tuliskan jenis, identitas dan status ketercapaian setiap luaran wajib dan luaran tambahan (jika ada) yang dijanjikan. Jenis luaran dapat berupa publikasi, perolehan kekayaan intelektual, hasil pengujian atau luaran lainnya yang telah dijanjikan pada proposal. Uraian status luaran harus didukung dengan bukti kemajuan ketercapaian luaran sesuai dengan luaran yang dijanjikan. Lengkapi isian jenis luaran yang dijanjikan serta mengunggah bukti dokumen ketercapaian luaran wajib dan luaran tambahan melalui Simlitabmas.
Draft jurnal internasional sebagai luaran dari penelitian ini sedang dipersiapkan dengan judul DNA Barcoding And cDNA Fragment Isolation of Actin-Encoding Gene From Philipine Tung (Reutealis Trisperma). Makalah tersebut rencananya akan disubmit di jurnal terindeks scopus (Heliyon, Q2). Selain itu, draft makalah yang lain dengan judul Foxtail millet (Setaria italica)-An Alternative C4 Model Plant : Potential Plant-based Bioenergy and Its Molecular and Biochemical Responses under Drought Stress, juga sedang dipersiapkan.
E. PERAN MITRA: Tuliskan realisasi kerjasama dan kontribusi Mitra baik in-kind maupun in-cash (untuk Penelitian Terapan, Penelitian Pengembangan, PTUPT, PPUPT serta KRUPT). Bukti pendukung realisasi kerjasama dan realisasi kontribusi mitra dilaporkan sesuai dengan kondisi yang sebenarnya. Bukti dokumen realisasi kerjasama dengan Mitra diunggah melalui Simlitabmas.
Penelitian ini tidak memiliki mitra
F. KENDALA PELAKSANAAN PENELITIAN: Tuliskan kesulitan atau hambatan yang dihadapi selama melakukan penelitian dan mencapai luaran yang dijanjikan, termasuk penjelasan jika pelaksanaan penelitian dan luaran penelitian tidak sesuai dengan yang direncanakan atau dijanjikan.
Tidak ada kendala yang berarti dalam penelitian ini
G. RENCANA TAHAPAN SELANJUTNYA: Tuliskan dan uraikan rencana penelitian di tahun berikutnya berdasarkan indikator luaran yang telah dicapai, rencana realisasi luaran wajib yang dijanjikan dan tambahan (jika ada) di tahun berikutnya serta roadmap penelitian keseluruhan. Pada bagian ini diperbolehkan untuk melengkapi penjelasan dari setiap tahapan dalam metoda yang akan direncanakan termasuk jadwal berkaitan dengan strategi untuk mencapai luaran seperti yang telah dijanjikan dalam proposal. Jika diperlukan, penjelasan dapat juga dilengkapi dengan gambar, tabel, diagram, serta pustaka yang relevan. Jika laporan kemajuan merupakan laporan pelaksanaan tahun terakhir, pada bagian ini dapat dituliskan rencana penyelesaian target yang belum tercapai.
Tahap selanjutnya yang akan dikerjakan adalah analisa in silico kekerabatan fragmen gen aktin R. trisperma dengan tanaman lain, khususnya dengan tanaman dari famili Euphorbiaceae.
H. DAFTAR PUSTAKA: Penyusunan Daftar Pustaka berdasarkan sistem nomor sesuai dengan urutan pengutipan. Hanya pustaka yang disitasi pada laporan kemajuan yang dicantumkan dalam Daftar Pustaka.
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2. Linhart, C., dan Ron Shamir. 2004. The Degenerate Primer Design Problem: Theory and Applications. Tel Aviv: Tel Aviv University.
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4. Lang, Michael., dan Virginie Orgogozo. 2011. Identification of Homologous Gene Sequences by PCR with Degenerate Primers. Methods in Molecular Biology. vol. 772.
5. Edgar R. 2004. MUSCLE: Multiple sequence alignment with high score accuracy and high throughput. Nucleic Acids Res. Vol. 32: 1792–1797.
6. Do, C.B., Mahabhashyam M. S, dan Brudno M. 2005. ProbCons: Probabilistic consistencybased multiple sequence alignment. Genome Research. Vol. 15: 330–340.
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21. Cazorla, E. R., Alfonso Andujar, Juan, J. R., Lindsay J. B., Antonio M. L. , Martin F. Yanofsky, dan Antonio Vera. 2015. 3’ Rapid Amplification of cDNA Ends (3’ RACE) Using Arabidopsis Samples. Bio Protoc. Vol. 5 (19).
DNA BARCODING AND cDNA FRAGMENT ISOLATION OF ACTIN-ENCODING GENE FROM Philipine Tung (Reutealis trisperma)
Nurul Jadid1*, Nur Laili1, Indah Prasetyowati1, Sri Nurhatika1, Hery Purnobasuki2, Tutik Nurhidayati1 1Department of Biology, Institut Teknologi Sepuluh Nopember, Surabaya-Indonesia
2Department of Biology, Universitas Airlangga, Surabaya-Indonesia
Abstract
Reutealis trisperma (Kemiri sunan) is a member of the family Euphorbiaceae which is currently being developed as biodiesel. This potential is influenced by genetic factors with different expression levels. One example to analyze the level of gene expression is through the Real-Time PCR method. For the development of the method, internal controls such as housekeeping-gene are needed, for example Actin gene. The objectives of this study were to identify R. trisperma molecularly, isolate and clone the actin gene fragment from R. trisperma which functions as an internal control in gene expression analysis, and to find out the relationship between the actin gene in R. trisperma and several species in the Euphorbiaceae famili. The identification of R. trisperma plants was carried out using DNA barcoding with candidate rbcL and matK genes. Isolation of actin cDNA fragments was carried out by the Reverse Transcriptase-PCR approach. Amplicon PCR results were then cloned on pTA2 plasmids. DNA sequences were obtained by sequencing then analyzed in-silico to determine the kinship relationship of cDNA of the actin gene R. trisperma. The results showed that the RtrbcL and RtmatK gene fragments were successfully isolated with sizes of 592 bp and 840 bp. The RtrbcL gene fragment provides more informative molecular phylogenetics data of R. Trisperma rather than the RtmatK gene fragment. The RtACT gene fragment was successfully isolated with a size of 986 bp. Analysis of phylogenetic relationships based on nucleotide base sequences showed that R. trisperma has the highest kinship level for the Vernicia fordii actin gene.
Foxtail millet (Setaria italica)-An Alternative C4 Model Plant :
Potential Plant-based Bioenergy and Its Molecular and
Biochemical Responses under Drought Stress
Choirotin Nisaa, Sri Nurhatikaa, Hery Purnobasukib, Nurul Jadida* aDepartment of Biology, Institut Teknologi Sepuluh Nopember Surabaya-Indonesia
bDepartment of Biology, Universitas Airlangga-Indonesia *Corresponding author : [email protected]
Abstract
Foxtail millet (Setaria italica) or locally named as Jewawut is one of the carbohydrate-source plants. Therefore, it is potentially used for functional food and biomass-based energy development. Among other varieties of millet, S.italica is easy to be cultivated in all over the world. Moreover, it has been also reported to be relatively tolerant to drought condition. In term of its function as potential nutritional food, this high protein cereal crop is also rich in dietary fiber, mineral, fat, vitamins and potential antioxidant properties. In addition, foxtail millet is also known as C4 model plants. Hence, engineering this C4 genomic properties might offer beneficial interest for accelerating their quality improvement. Characteristics of several accessions and varieties of foxtail millet are also described in this review to give a comprehensive overview concerning their potential uses. Finally, some aspects concerning its responses under drought stress condition are also shared for giving an update perspectives to exploit this plant in neglected soils.
1. Introduction
Setaria italica or commonly known as millet is generally seen as the main crop in many developing countries that is cultivated on marginal agricultural land in areas with low rainfall. In the region, the more common cereals cannot grow and millet in various forms, which includes more than 100 wild and cultured species, providing most of the energy and protein needs for millions of people, especially in sub-Saharan. Africa and parts of Asia (Ermawar et al. 2015). Setaria italica is a panacoideae family that has been proposed as an ideal model system to accelerate the discovery and characterization of plant genes that control agronomically important traits. Phylogenetically, Setaria italica is related to economically important plants as well as bioenergy such as Zea mays, Sorghum bicolor and Panicum virgatum (Tang et al. 2017). Setaria italica has a relatively small diploid genome (~ 510 Mb), has a short annual life cycle (50-90 days) and is a C4 plant (Diao et al. 2014; Layton and Kellogg 2014; Tang et al. 2017) and can produce more than 10,000 seeds per plant (Brutnell et al. 2010; Ermawar et al. 2015). Morphologically S.italica or Foxtail millet is an annual grass with slender stems, upright, leafy with a height of 170 cm (Yulita 2018). Inflorescence consists of a narrow panicle that often nods at the top and looks like a spike because of its short branches. Spikelet (one pistill per spikelet) is packed, and with many strong feathers, the panicle looks like a foxtail, and hence the name refers (Lata, Gupta, and Prasad 2013) (Fig. 1). This plant has been genetically transformed successfully through Agrobacterium media (Brutnell et al. 2010). Thus, Setaria italica can be used as a model plant to study complex physiological and molecular basic mechanisms, especially in cereal plants. At the present, Arabidopsis thaliana still functions as a standard for assessing many abiotic developments, illnesses and stress responses. But it does not rule out Setaria italica also has the opportunity to define differences in the structure and function of genes. Besides Setaria italica can be used as a model of C4 plants to overcome the most pressing agricultural problems in the world, namely abiotic stress tolerance. This review focuses on several aspects of its response in drought conditions as well as being shared to provide a current perspective to exploit this plant in neglected soils.
Figure 1.A, Habit, foxtail millet variety Yugu1 (scale = 6 cm). B,Yugu1 mature inflorescence (scale = 4 cm). C, Green millet variety A10 (scale = 4 cm. this picture was adopted from (Doust et al. 2009).
Genetic diversity of Setaria italica
Testing genetic diversity in plant germplasm is an important aspect of plant breeding. Accurate measurement of the level and pattern of genetic diversity is important to identify diverse breeds to make crosses so as to produce the development of superior cultivars. The Andropogoneae tribe includes corn, sugar cane, and sorghum. Plants with the genus Setaria are closely related to the switchgrass of biofuel feedstocks and guinea grass (Fig.2). The Setaria genus consists of about 125 species that are widely distributed in tropical climates throughout the world (Lata et al. 2013). Each genus has diverse phenotypic properties among species. Based on the morphology of flowering Setaria are classified into two species of S.pumila and S.italica. S.italica species consist of two subspecies viridis and italica. Italica subspecies are classified into three races namely aristate, fusiformis and Indica (Upadhyaya et al. 2008). In Indonesia, S. italica consists of accessions of yellow Polman, Red Polman, Sweet Gambir and Red Buru, each of which has morphological characters that appear from different seed colors (Fig.3). However, in Indonesia the four accessions have not been studied more deeply. Some S.italica cultivars are very tolerant of drought, which is used as the main crop in arid regions in North China with low rainfall conditions. Setaria species deal with abiotic stress by using NADP-Malic enzyme subtype C4 photosynthesis system to repair carbon. In addition, C4 grass has a
Figure 2. Phylogenetic relationships of grass family members. The tree is adapted from (Li and Brutnell
2011). Branch length does not reflect evolutionary divergence time.
Figure 3. Setaria italica seed color differences from each accession
superior photosynthate translocation and water distribution system structurally by developing networks that are denser than longitudinal and small transverse veins. Setaria has high venous density and kranz anatomy which helps concentrate CO2 in the bundle bundle cells. So as to minimize photorespiration and prevent energy loss (Vivitha et al, 2015). A large number of S. italica's accessions are widespread geographically and occupy diverse ecological niches. Thus there is an opportunity for mapping diversity where there is a possibility that gene flow has formed between wild accession and cultivation. Besides functioning as an excellent model for C4 photosynthesis, this grass can also be used to study abiotic stress tolerance as well as models for bioenergy feedstocks.
The increasing demands for food have posed a serious challenge to agriculture. In general, crops such as rice, wheat and corn are the main cereals in the world of food, followed by wheat, sorghum, millet and oats. Other cereals are known as millet minor. But millet is an important staple in a large number of countries in the semi-arid tropics, where food crop cultivation is mainly limited by low rainfall and poor soil fertility. Therefore millet is needed as a food crop in rainfed agroecosystem or with neglected soil conditions.
Some S.italica cultivars are very drought-tolerant where per 1 gram of dry biomass, corn plants need 470 grams of water, wheat 510 grams of water, and S. italica 257 g (Li and Brutnell 2011).
As a food source that is rich in nutrition, barley is recommended for consumption by children and adults. This is because barley is rich in protein, fiber, minerals and vitamins, and hence can be referred to as nutricereals (Upadhyaya et al. 2011). Foxtail millet or Jewawut (S.italica spp. Italica) has been consumed in several regions such as Sulawesi, Maluku and East Nusa Tenggara, but in other areas, Java is known as bird feed because it is rich in fiber (Yulita 2018). As a source of bioenergy, Jewawut has excellent potential for use as a bioenergy feedstock from its lignocellular biomass (Muthamilarasan et al. 2015). In addition, genes involved in lignocellulosic biosynthesis are also reported to play an important role in the abiotic stress response. Based on research (Guerriero, Legay, and Hausman 2014) has successfully identified nine expression profiles of cellulose synthase in stems, roots, and leaves in alfalfa plants with different abiotic pressure conditions, cold, heat and salt stress). In addition, various studies through a systematic approach have been used to identify gene families involved in cellulose (CesA / Csl), callose (Gsl) and monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD) ) the results showed that phylogenetic analysis of the identified proteins showed that monolignol biosynthetic proteins were very diverse, whereas the CesA / Csl and Gsl homologous proteins with rice and Arabidopsis (Muthamilarasan et al. 2015). The production of environmentally friendly biofuel so far has also become an issue that must be developed, one of which is with the help of mycorrhizae and the use of plant growth enhancing bacteria (PGPB). It has been reported that the combination of PGPB and mycorrhizae can increase biomass and total sugar in Setaria italica plants (Dhawi, Datta, and Ramakrishna 2017).
Genetic Engineering in Setaria
In vivo funtional analysis is very important especially to study the molecular mechanisms of abiotic and biotic stress tolerance so that it can be used as a basis for plant breeding. Several approaches have been taken to see the response of plants that are tolerant of abiotic or biotic stress, namely the molecular response through gene expression and physiological responses through secondary metabolite compounds in the plant system.
Metabolomic known to play a significant role in the study of biology in plants with choking conditions. In response to abiotic stress, plants undergo changes in primary and secondary metabolic compounds that can be identified and analyzed physiologically and molecularly. Several studies that have successfully analyzed changes in metabolites can be observed in Table 1. Transcriptomic studies using leaf samples treated with drought for 6 days on the Setaria italica plant successfully identified 3066, 1895, and 2148 genes that were expressed differently (DEG) in M79, E1 and H1 were compared compared to controls (Shi et al. 2018). According to (Tang et al. 2017), the results of the analysis with QTL related to drought tolerance have identified 20 candidate genes that contribute to germination and drought tolerance of early S.italica seedlings. so based on that further study related to differences in S.italica genotype in responding to drought is very important to do to improve genetic tolerance to drought in Poaceae plants.
Table 1. Differential Regulation of Important Metabolites in C4 Plant Response to Drought stress Class of
metabolites Metabolite Plant Species
Regulatio
n Function References
Polyols
Organic
acid Oryza sativa Up
Precursor for many compounds
(Farooq et al. 2010) Spermidine
Oryza sativa Down Membrane
integrity, signal transduction
(Do et al. 2013)
Sugar alcohol
Myoinosito
l Zea mays Up Osmotic adjustment
(Miguel et al. 2016) Amino acid Proline Arabidopsis thaliana Up Protein structure stabilization, promotes activity of antioxidant enzymes. (Nemat Alla, Khedr, and Nada 2012)
Zea mays Up (Lugan et al.
2010) Setaria italica Up (Paul Ajithkumar and Panneerselvam 2013)
Tyrosine Zea mays Up Osmoprotectant,
cell signaling
(Nemat Alla et al. 2012)
Valine Zea mays Up Protein amino acid (Muscolo et al.
2015) References
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