KHOA HOC CONG NGHE
IMGHIEIM C O U PHAIM L A P PRDIVIOTER asSWEET14 V A T H I E T K E C A U TRIJC gRIXIA TAIXIG CUOIMG K H A IMAIMG
K H A I M G BEIMH B A C L A CIJA GIOIMG L U A T B R B B B
Phimg Thi TTiu HuongS Nguyen Duy PhuongS Phgm Xuan Hpi^
TOMTAT
OsSWEET14\k ho gen ma hoa cac protein vdn chuyen saccrose, co lien quan den ca che xam nhi^m cua vi khuan bac la o thuc vat. Trong nghien cuu nay, promoter OsSWEETll da duoc phan lap tii DNA tong so ciia gidng lua chii luc TBR225 mdn cam voi vi khuan gay benh bac la Xanthomonas oryzae pv. oryzae (Xoo).
Trinh tu DNA phan lap duoc co kich thuoc 1392 bp, chtra 4 trinh tu bam dac hieu (EBE) cho protein ttet loai III (TAL effector) cua vi khuan bac la Xoo, bao g6m TaiC. Tal5, PthXaS va AvrXa7. OsSWEETM ciia gidng lua TBR225 gidng lan luot 99% va 100% so voi OsSWEETM ciia gidng liia Japonica (Niponbare, ma sd AP014967 1) va Indica (Shuhui498, ma sd CP018167.1) da duoc cong bd. Dua tren trinh tti DNA phan lap duac, 3 cau tnic gRNA (guide RNA) da duoc thiet ke voi mue dich chinh sua vi tti bam dac hieu ciia TAL effector tren promoter OsSWEET14 va bat hoat hoan toan gen OsSWEET14 bang cong nghe CRISPR/CAS9 (Clustered regulariy interspaced short palindromic repeats/CRISPR associated protein 9) nhdm tang cuong kha nang khang benh bac la ciia gidng liia TBR225. Nghien ciki nay la co so cho viec tao ra cac gidng liia nang suat cao co kha nang khang benh bac la bang cong nghe gen cr Viet Nam.
^ii\ih6a: Bac la. CRISPR/CAS9, OsSWEET14, TAL effector, Xanthomonas oryzae.
I.DATVANDE
Gidng lua TBR225 duge Cong ty Gidng cay trdng Thai Binh chgn tao. Id mot trong nhung gidng ehii lire ciia Viet Nam do ed nhilu tinh trang quy nhu tinh thich ung rong, chdng ehiu voi didu kien thdi tiet bat thuan vd sau benh hai khd tdt, nang sudt trung binh dat 60 - 75 ta/ha... Nhuoc diem Idn nhat cua TBR225 la kha nhay cam vdi vi khuan bac la Xanthomonas oryzae-^v. oryzae (Xoo), bi thiet hai trung binh hang nam 10-20% san luang[101.
Xoo gay benh tren cay iiia thong qua he thdng protem tiet loai III la TAL (ti-anscription activator- like) effector. TAL effector bdm vao trinh tu dich dae hieu (effector binding element - EBE) tren viing promoter cua gen nhidm (5gene), bao gdm cde gen SWEET, ti-ong he gen td bdo ehu va hoat hod gen dich de thuc day qua ti'inh lay nhilm eua vi khudn [7]. Cdc thdnh vien cua hg gen OsSWEET ma hda cho cdc protein van chuyen sucrose tir nhu mo tdi maeh ddn ciia mo hbe [5]. Mot sd gen tiiuoe nhom III ciia hp gen SWEET, bao gdm OsSWEETll, OsSWEET13va OsSWEETMda duoc chung minh la gen "nhilm" (gen S- susceptibility gene) ddi vdi Xoo [2,13]. Nghien cuu he gen ciia mot vai gidng lua eho
thay dot bidn mat 18 bp tren promoter OsSWEETM ^^^^^^^er/gen OsSWEETU. Kit qua thu dupe s tiln d l eho nghien ciru tao gidng lua cd khd nang khdng benh bac Id, cho nang sudt cao bdng cong ' Vien Di iruygn Nong nghiep nghe CRISPR/CAS9.
Enmd: [email protected]
cd hen quan tdi tinh khdng hon 50% sd ehung vi khuan Xoo eua chau A va chau Phi (da bilt) [7].
Tuong tu, bdng each gay dot biln nhan tao tren vimg promoter eiia OsSWEETU, Blanvillam-Baufume cung da tao dugc ddng liia khdng mpt sd ehung vi khuan bac la mang TAL effector tdc dSng vao gen OsSWEET14{2]. He thdng CRISPR/CAS9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) dang ngdy edng phat trien va duac iing dung nhu mot bo cong cu chinh sua he gen trong sinh hpc phan tii' va t l bao de tao eac dot biln ed chii dich nham dieu chinh ehiic nang eiia gen dieh, dua tren tinh dac hieu eua ede trinh tu gRNA (guide RNA) [4]. CRISPR/CAS9 cho phep thay ddi tinh trang cay trdng trong khi khong san pham tao ra khong mang DNA ngoai lai.
Do dd eong nghe nay rat phii hap voi cdc nghien cuu cdi tiln cac tinh trang d lua.
Vdi mue tieu eai thien khd nang khang benh bac la eua gidng lua chu luc TBR225, ddng thdi lam phong phii dir heu ngan hang gen cac gidng liia Viet Nam, da tiln hdnh phan lap va nghien euu trinh tu promoter/gen OsSWEETM cua gidng lua TBR225 (goi tdt Id OsSWEET14-TBl^ vd xde dmh mot sd trinh tu gRNA dac hieu cho viec ehinh sua
42 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 12/2018
KHOA HOC C d N G N G H l
2 . VAT UEU VA PHUONG PHAP NGHIBU CUU 2.1. Vat ueu
Gidng Ilia TBR225 do Tdng Cong ty Gidng cay trdng Thdi Binh cung cdp; ehiing vi khuan E. coh DH5a dugc mua tir Hang Thermo Fisher Scientific
(My).
Cdc oligonucleotide dimg cho PCR nhan ban OsSWEET14-TBR duoc thilt ke dua ti-en ti'inh tu da dupe cong bd tren GenBank (AP014967.1) va dat mua tii Hang Sigma (My).
2.2. Phuang phap
2.2.1. Tich chiet DNA tdng sd tu eay lua xu li stress
DNA tdng sd eua cay chuyin gen dugc tach ehiet theo phuong phap eiia Doyle et al. (1990) [6], su dung dung dieh CTAB 2%. Mdu mo thuc vat tuai (100 mg) duac nghidn trong nito ldng, sau do duac bd sung 500 pi dung dich (HAB 2% (cd chira ARNase 40 mg/ml) va ly tam d toe dp 13.000 vong/phut de thu dieh ndi. 500 pi hdn hgp phenol : chloroform : isoamyl (25 : 24 : 1) duge bd sung vao dung dich d l k i t tiia protein. Hdn hop sau duac ly tam tdc do 13.000 vdng/phut d l thu DNA tinh sach..
2.2.2 Phan lap promoter GsSWEETlf-TBR Promoter cua gen OsSWEETM^TBR duoc phan lap tir DNA tdng sd eua eay lua non bdng ky thuat PCR [11], sir dung hai mdi SW14-Fw (5'- CATGCATATGCGTTGGGTTC-3') va SW14-Rv (5'- CTAGGAGACCAAAGGCGAA03'). San pham PCR 1392 bp duge tinh sach bdng bo kit GenJET-TM Gel Extraction cua Hang Thermo Fisher Scientific.
2.23 Nhan ddng promoter OsSWEET14-TBR Promoter OsSWEET14-TBR nhan bdn bdng PCR dugc nhan ddng bdng bo kit pGEM-T Easy theo quy trinh di kem cua Promega. Vector tai td hgp duac biln nap vao vi khuan E. coli DH5a; thi bien nap duoc sdng Ige bdng phuong phdp PCR true tiep khuan lac vdi cap mdi T7/SP6. Plasmid dugc tach ehilt tir vi khuan E. coh bang bp kit GenJET-TM Plasmid Miniprep ciia Thermo Fisher Scientific. San pham plasmid tdi td hgp dugc kiem tra bdng ki thuat PCR vai 2 cap mdi SW14-Fw/SW14-Rv va T7/SP6 va bang phan ung cdt gioi han vdi cae enzyme EeoRI, Bglll va Ndel.
2.2.4. Phan tich trinh tu OsSWEET14-TBR
Trinh tu gen dugc xdc dinh bdng thilt bi tu ddng AB13100 theo phuang phdp cua Smitii et al (1986) [12], sir dung hai mdi T7 vd SP6 eho phan ung PCR gidi trinh tu. Ket qua giai trinh tu duge xu li bdng phdn mIm BioEdit 4.0. Trinh tu DNA sau khi xii \i duge so sanh vdi ca sd dir heu tren Gene Bank vd phan tieh bdng phdn mem Genetyx 4.0.
2.2.5. Thiet ke trinh tu gRNA dac hieu cho OsSWEETM-TBR
Trinh tu gRNA dac hieu eho promoter OsSWEETM-TBR dugc tiiiet k l bang phan mem
CRISPR MultiTargeter (http://www.multicrispr.net/). Hdm luong GC cua
ede gRNA duge phan tieh bdng phan mIm Genetyx 4.0. Cdu tnic bae II eiia cdc gRNA dugc phan tich bdng pham mIm Mfold 2.3 (htip://unafold.rna.albany.edu/). Doan DNA gidng/gdn gidng voi cdc gRNA trong he gen liia duac xac dinh bang phan mIm CCTop (http://erispr.cos.uni-heidelberg.de). Trinh tu he gen liia duoe tham chieu tir ea sd dii lieu tren ngan hang gen Ensembl Plants (http://plants.ensembl.org/). Doan gRNA dac hieu duac lua chpn dua tren vi tri tren promoter/gen OsSWEETM-TBR, hdm lupng GC, kha nang hinh thdnh va duy tri eau true bac II, sd lupng vd vi tri doan DNA gidng/gan gidng xuat hien trong he gen liia, vi tri va sd lugng nucleotide sai khae tren gRNA [8].
3. KET QUA NGHIEN CUU
3.1. Phan l§p promoter OsSWEET14-TBR eua gidng Ilia TBR225
Vimg promoter ciia gen OsSWBET14-TBR dugc nhan ban bdng PCR voi khuon Id DNA cua mau liia non, sii' dung cap mdi dugc thief ke dua vao trinh tu nucleotide eiia gen OsSWEETM duoc cong bd tren Genbank. Kit qua dien di tren gel agarose 1% eho thay san pham PCR ehi xuat hien mot bang DNA duy nhdt cd kieh thudc xdp xi 1,4 kb tuong ting vdi kich thudc li thuylt eiia doan promoter OsSWEETM-TBR (1392 bp) (Hinh 1, gieng 1). Nhu vay, doan DNA mong mudn da dugc phan lap thdnh eong til' DNA tdng sd eua mdu lua non.
SWEET la hg gen ma hda cho cde protein van chuyen dudng, duge eho la cd hen quan den ea ehe xam nhilm eua vi khuan Xoo gay benh bac Id thong qua qud trinh giai phdng phan tii dudng vdo gian bdo
NONG NGHIEP VA PHATTRIEN NONG THON - KY 1 - THANG 12/2018 43
KHOA HQC CdNG N G H £
(apoplast) va cung cdp dinh dudng eho vi khuan. Mot so gen nhu OsSWEETll, OssWEET13 vd OsSWEETM da duoc xdc dinh la dich tae dong ciia mot sd TAL effector va hoat dgng nhu gen "nhilm"
( 5 gene) ddi vdi Xoo d lua [2]. Trong nghien eiiu truoc day ve vai ti'd ciia gen OsSWEETM ddi voi c a e h i xam nhidm ciia Xoo tren gidng lua IRBB13, doan DNA ddi 341 bp ndm ti-en promoter OsSWEETM cung da duae phan lap d l nghien cuu chu'c nang
[ 13]. Trong nghien cuu ndy, trinh tu promoter ddy du va mot phdn ma hod tren gen OsSWEETM cua gidng lua TBR225 da dugc phan lap bdng PCR tij DNA tdng sd de phan tieh trinh tu nucleotide nhdm du dodn vai trd eua gen OsSWEETM ddi vol tinh mdn cam vdi benh bac la cua lua TBR225, ddng thdi phuc vu nghien ciiu chinh sua promoter/gen OsSWEETM sau nay.
Hinh 1. Phan lap promoter OsSWEET14-TBRtii he gen lua bdng ki thuat PCR Gilng M: thang DNA 1
kh; gilng 1: san phdm PCR
3.2. Nhan ddng doan promoter OsSWEET14' TBRvao vector pGEM-T
San pham PCR nhan ban doan promoter OsSWEETM-TBR tir DNA tdng sd duge tinh sach, sau dd duac ghep ndi vao vector nhan ddng pGEM-T va bien nap vdo t l bdo kha hien E. coh chimg DH5a.
The biln nap Id cde khuan lac E. cob man trdng xuat hien tren moi trudng chpn loc dugc sang Igc de Idem tra su ed mat eua plasmid tdi td hap mang doan promoter OsSWEET14-TBRhkig phuong phdp PCR true tilp khuan lac vdi cap mdi vector Cr7/SP6). Mdt khuan lac trdng ed k i t qud PCR duong tinh sau do dugc chgn ngdu nhien de nuoi cay, tdch chief DNA plasmid vd kilm tra bdng phuang phdp PCR va cat bdng enzyme gidi han (Hinh 2 A va B).
Ket qua dien di tren gel agarose 1% tren hinh 2 cho thay sdn pham PCR tir plasmid vdi cap mdi SW14-Fw/SW14-Rv dae hieu cho doan promoter OsSWEETM-TBR, cho mot bang ed kieh thudc
khoang 1,4 kb (Hinh 2A, gieng 2), ti'ong khi san pham PCR vai cap mdi T7/SP6 dac hieu eho vector pGEM-T (ed khodng cdch 186 bp ti-en pGEM-T) eho mpt bang DNAcd kieh thuoc xap xi 1,6 kb (Hinh 2A, gieng 4), phii hgiJ vdi kieh thudc li thuylt eua doan DNA cdn nhan ban.
Tuong tu, k i t qua dien di sdn pham cdt gidi han plasmid tai td hgp bdng £coRI (doan DNA duac chen vdo giira hai vi tri nhan biet eua Ecd&\ tren vector pGEM-T) (Hmh 3B, gilng 1) hay Ndel (ed 1 vi tii nhan bilt d Nu thii 6-11 tren promoter OsSWEETM TBRva 1 Vl tri: khde ti-en vector pGEM-T) (Hinh 3B, gilng 2) ddu cho hai bang DNA cd kich thudc Idn luat khoang 3,0 kb tuong ung vdi bp khung vector pGEM-T va 1,4 kb tuong iing vdi doan promoter OsSWEETM-TBR dung nhu tinh toan h thuyet.
Ngoai ra sdn pham cdt gidi ban ddng thai bdng Ndel va BgAl (co 1 vi tri nhan bilt d Nu thii 1331-1336 tren promoter OsSWEETM-TBR) cung thu dupe cdc bang DNA diing vdi tinh toan theo ly thuylt, trong dd doan OsSWEETM-TBR bi eat thdnh 2 manh cd kich thudc khoang 1,3 kh vd 0,1 kb (Hinh 3B, gilng 3). Cde ket qua thu duge nay cho phep khdng dinh chdc ehdn hon viee nhan ddng thanh cong doan promoter OsSWEET14-TBRvao vector pGEMT.
M 1 2 3 j tb " •• 2 3
Hinh 2. Kilm tra vector tdi to hpp pGEM/0sSWEET14-TBR San pham PCR va citgidi han dupe dien di tidn gel agarose 1%. (A) PCR plasmid tii td hpp; gieng 1- 2: PCR vdi cap mdi SW14'Fw/SW14-Rv; gieng 3-4:
PCR vdi cap mdi T7/SP6; gidng 1 vi 3: ddi chiing Mi (khdng cd DNA khudn); gieng 2 vi 4: khudn la pGEM/OsSWEETM-TBR. (B) Cit gidi han plasmid tii td hop bing enzym cit gidi han; gieng 1: san pham eit gidi han bang EeoRI; gidng 2: sin phim cat gidi ban bing Ndel; gieng 3: sin pham cat gidi han bing Ndel/BglH; gieng 4: vector pGEM/0sSWEET14-TBR nguyen bin; gidng M:
thang chuan DNA 1 kb.
44 NONG NGHIEP VA PHATTRIEN NONG THON - KY 1 - THANG 12/2018
KHOA HOC CONG NGHE
Trong hdu het cae nghien ciru ve promoter da eong bd horde day, doan DNA quan tam thudng dugc phan lap va nhan ddng vdo vector nhan ddng trudc khi dua vdo vector bilu hien nhdm phuc vu viec nghien cuu chiic nang [9]. Dua tren cdc trinh tu tham khao tren ngan hang gen, ede tae gid da sii dung ky thuat PCR de nhan ban doan DNA ddi khodng 500 - 2000 hp ndm phia trudc vi tri ciia bp ba ma md dau nhdm thu duge doan promoter mong mudn (1). 0 nghien euu nay, doan DNA nam tir vi tri
(-1343) din (+52) ciia gen OsSWEETM da duae nhan hdn tir DNA tong sd eiia gidng lua Bde thom 7 vd ddng hoa vao vector pGEM-T. Sdn pham nhan ddng la ngudn nguyen lieu cho todn bp cde fhi nghiem nghien ciru dac dilm chirc nang eua promoter OsSWEETM sau ndy.
3.3. Phdn tich tiinh h; OsSWEET14'TBR ciia gidng liia TBR225
De khdng dinh chmh xde doan DNA phan lap dupe Id promoter OsSWEETM, vector tdi td hop pGEM/ OsSWEETM-TBR dugc giai ti-inh tu nucleotide va phan tich bdng phdn mIm BioEdit. Ket qua so sanh trinh tu nucleotide cho thdy doan promoter OsSWEETM phan lap duae tir gidng lua TBR225 cd ehilu ddi 1392 hp, gidng 99% so vdi trmh tu promoter OsSWEETM eua gidng liia Niponbare Gaponica, ma sd AP014967.1) va gidng 100% so voi trinh tu promoter OsSWEETM giong lua Shuhui 498 (Indiea, ma sd CP018167.1) da duge eong bd tren GenBank. Trinh tu phan lap duoc bao gdm vimg promoter ddi 1343 bp va mdt doan ddi 52 bp mang trinh tu EXON I cua gen OsSWEETM. Doan promoter OsSWEETM phdn lap dugc cung ehiia trinh tu TA Box dac trung d vi tri -259. Dac hiet, tren vimg promoter OsSWEETM ciia lua TBR225 ed 4 chiia trinh tu DNA ^ e u td dilu hda c/s) hen kit dac hieu vai protein TAL eua mdt sd ehiing vi khuan Xoo da duge cong bd [7], bao gdm TalC, Tal5, PthXo3 va AvrXa7(Hmh3).
Nhu vay, vi khuan Xoo khi xam nhilm vao te bao Ilia TBR225 cd t h i tilt ra cde protem TAL hoat dong nhu nhimg nhan td phien ma; protein TAL se lien kit vol cdc EBE tren vimg promoter cua ede gen van chuyen dudng (trong dd cd OsSWEET14), hoat hda gen de bilu hien ra cde protein SWEET van chuyen dudng phuc vu cho qud trinh sinh trudng ciia vi khuan. Dua tren dii lieu day du ve trinh tu nucleotide Ciia gen mue tieu, sii dung cong nghe gay dot bien
chinh xae tai cdc vi tin EBE ed the ngan ehan/giam dpc tinh cua vi khuan Xoo h-en gidng liia TBR225.
Ngodi ra, viec gay dot bien dich khung (them/bot nucleotide) tren vimg EXON I de bat boat hodn toan gen (knock-out gene) OsSWEETM eung la mot hudng nghien eiiu d l tao ra gidng lua TBR225 khang hae la.
Hinh 3. Phan tich tiinh ti; promoter OsSweetl4-TBIL Hop TATA box dugc ddng khung. Vi hi cdc EBE va
EXONl dirpc ddnh dau bang mui ten 3.4. Thilt k l cau tnic gRNA chinh sua promoter/gen OsSWEET14 cua gidng liia TBR225 hdng cong nghe CRISPR/CAS9
He thdng chinh sira gen CRISPR/Cas9 hoat dgng dua tren kha nang edt chinh xde DNA soi doi tai vi tri mong mudn cua phiic he protein-RNA CR1SPR/Cas9, trong dd bao gdm hai thdnh phdn chinh: (1) protein Cas9 eo hoat tinh endonuclease va (2) phan tu sgRNA (smgle guide RNA) eo vai trd ddn dudng eho phiie he din diing vi tri DNA can edt.
Tinh dae hieu phiic he CRISPR/Cas9 dugc quylt dinh hdi doan trinh ty ddi -20 nucleotide (crRNA) tren phan tir sgRNA [4]. Dl phuc vu nghien ciiu chinh sira promoter/gen OsSWEETMTBRhkng he thdng CRISPR/Cas9, ti-inh hi' crRNA duoc thilt k l nhdm gay dpi biln tai eae vi tri EBE tren promoter OsSWEETMhoac trin EXON I.
Bdng phdn mem CRISPR MultiTargeter, 119 trinh tu (20 nu) tren promoter cua OsSWEETM-TBR da duae xae dinh ndm phia tiirde vi tri nhan bilt bao thu PAM (protopaeer adjacent motif-NGG), bao gdm 57 trinh tir thuoc spi DNA {+) va 62 trinh tu thupe sgi
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 12/2018 45
KHOA HOC C 6 N G NGHi
(-) (Dir lieu khong dirge trinh bay). Trong do, 2 trinh tu crRNA CO diem cat tai trinh tu TalC, 8 trinh tu crRNA CO dient cat tac dong den ca ba trinh tu
Bang 1. B j c diem c^c gRNA chinh siia OsPSCS-BC
AvrXa? EBE, PthXoS, Tal5; 6 trinh tu crRNA CO diem cdt tac dong den Exon I (Bang 1).
Ten gRNA-1 gRNA.2 gRNA-3 gIiNA-4 gimA-5 gRNA-6 gRNA-7 gRNA-8 gRNA-9 gENA-10 gRNA-11 gENA-12 gRNA-13 gRNA-14 gRNA-15 gRNA-16 gRNA-17
Trinli tir crRNA 5'- TGCTGACATGCATGCCCTrA.3' 5'- AGGGCATGCATGTCAGCAGC-3' 5'- GAGGGGGTrrATATAGTGCT-3' 5'- TATATAAACCCCCTCCAACC-3' 5'. GCTrAGCACCTGGTrGGAGG.3' 5'- AGCTTAGCACCTGGTTGGAG-3' .5'- GAGCTTAGCACCTGGTTGGA-3' 5'- TGAGCTTAGCACCTGGTTGG-S' 5'- TGATGAGCTTAGCACCTGGTS' 5'- GGCTrGATGAGCTrAGCACC-3' 5'. CTTGAGTTTGCTlTGCTrGA-S' 5'- CAAGATCTTGCTGCAATGGC-3' 5'- GCATGTCTCTTCAGCATCCC-3' 5'- CATGTCTCTTCAGCATCCCT-3' 5'- CATCCCTGGGCCTTCGCCTT-3' 5'- AGACCAAAGGCGAAGGCCCA-3' 5-- GAGACCAAAGGCGAAGGCCC-3'
GC 50 60 45 45 60 55 55 55 50 55 40 50 ,55 50 65 60 65
TBP 8 7 9 0 8 8 8 8 12 9 7 7 2 4 9 10 10
CB P 0 4 7 0 6 6 6 6 6 4 4 3 0 2 5 4 4
IBP 4 3 0 0 2 2 2 2 0 5 0 4 2 2 0 0 0
DSL SLl SI,1 SLl SLl
•
SLl SLl SLl SLl SLl SLl SLl SLl
•
SLl SLl SLl
On target
29.5 51.4 51.5 57.1 50.6 51.9 50.3 40.3 48,4 57.9 27.8 44 26.8 50,8 32 59.5 N/A
Off target
48.4 47.6 39.8 49.2 48.1 48.7 49 48.(LJ 49.6 48.4 46.1 48.4 48.6 48.6 49.3 48.7 48.7
Dicli tac dpng TalC TalC AvrXa7, PthXo3 AvrXa7, PlhXo3,Tal5 AvrXa7, PthXo3, Tal5 AvrXa7, PthXo3, Tal5 AvrXa7, nhXo3, Tal5 AvrXa7, RhXoS, Tal5 AvrXa7, RhXo3, Tal5 AvrXa7, PthXo3, Tal5 AvrXa7, PlhXo3, Tal5 Exon I Exon I Exon I Exon I Exon I Exon I
Ghi chu: (TBP) tdng sd cap baza; (CBP) sd cap baza hen tue; (IBP) sd cap baza tiong eau tide ciia crRNA; (DSL) vdng loop bi phi vd eau tide; (RAR) Stem loop Repeat and Anti-repeat Region, (SLl, 2, 3) Stem loop 1, 2, 3; (*) khdng anh hudng den cau true vdng loop; (TalC, AvrXaZ, PthXo3, Tal5) Cie EBE tidn promoter OsSWEET14-TBR; (On target) Didm du doin kha nang nhin biet dung vi tii dich (tuO-100); (Off
target) Diem du doan kha nang nhan biet sai vi tri dieh (tuO-100).
bao, gRNA phai co hdm lugng GC trong trinh tu
\ r^
i-i-^-A4./
Hinh 4. Cau hnic bac II ciia gRNA-10 Md hinh cdu tnic bae II duac du doin bing phin mdm Mfold2.3mang vimg crRNA (CRISPRRNA) va cae eau tide vdng loop: Stem loop RAR (repeat and anti-repeat region), Stem loop2 vi Stem loop3.
Bi dam bdo khd nang duy tri hoat tinh vd tinh dae hieu eua phuc he CRISPR/CAS9 trong nhan t l
crRNA tir 30-80%, ed kha nang hinh thanh eau tnic bae II dn dinh (duy tri cdu tnic vdng loop RAR, SL2 va SL3), tdng sd cap baza CTBP-total base pairs) < 13, sd cap baza hen tuc (CBP-consecutive base pairs) <
8, sd cap baza ben trong cau tnic crRNA (IBP- intemal base pairs) < 7 (8]. Kit qua phan tich bdng phdn mem Genetyx 4.0 va Mfold 2.3 cho thay cdc gRNA deu dam bdo viec hinh thdnh phiie he CRISPR/CAS9 dn dinh eo hoat tinh trong nhan te bao thuc vat (bdng 1, hinh 4).
Ben eanh nhimg uu diem ve tinh don gian, thuan tien vd kha nang img dung Idn, he thdng ehinh siia gen CRISPR/Cas9 co nhugc diem so vai eae he tiidng ehinh sua gen khdc CTALEN, ZFN...) dd la tinh dae hieu, do sgRNA chi nhan bilt 20 nucleotide va Cas9 vdn cd the hoat dpng dii trinh tu DNA dich co mpt vdi nucleotide sai khac so voi crRNA [4]. Nhdm giam thieu kha nang nhan biet sai vi tri (off-target) ciia phiic he Cas9-gRNA, trinh tu cua cae crRNA tiep tuc duge phan tich bdng cong eu CRISPR trong phdn
46 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 12/2018
KHOA HOC C 6 N G NGHE
mIm Benchhng (https://benchlmg.com) de du dodn tinh dac hieu cua eac trinh tu crRNA. Kit qua phan tieh (Bang 1) eho thay eac hinh tu gRNA2, gRNA4, gRNA6, gRNAlO va gRNA 16 cd dilm dac hieu cao nhat.
Tiep theo, trinh tu vd vi tri chinh xde ciia cde doan DNA gidng/gdn gidng vdi 5 crRNA ndy cd trong he gen lua (http://plants.ensembl.org/) tilp tuc duge xdc dinh hdng phdn mIm CCTop (http://erispr.cos.uni-heidelberg.de). Kit qua phan tich eho thay gRNA-2 (chinh sua vi tri cua TalC) ed 1 trinh tu DNA tuong ddng trong he gen liia vd gRNA- 16 (ehinh sira vi ti-i ciia EXON I) cd 11 trinh tu DNA tuong ddng. Tat cd cdc trinh tu DNA tuong ddng voi hai crRNA ndy diu khSng ndm trong vung ma hda
cua gen vd cdch xa vi tri eua exon gdn nhdt. Ddi vdi 3 crRNA ehinh sua 3 EBE AvrXa7, PthXo3, Tal5, gRNA-4 va gRNA-10 cd 1 trinh tu DNA tuong ddng khong ndm tren viing gen ma hda, nguac lai gRNA-6 cd trinh tu DNA tuong ddng nam tren viing ma hda (exon) eiia mot gen ma hda protein chua hilt ehuc nang nen ed kha nang gay anh hudng din hoat ddng chiic nang eua t l bdo nlu xay ra dot hien sai vi tri mong mudn. Ben eanh do, mac du trinh tu DNA tuong ddng vai gRNA4 va gRNA-10 diu ed 4 Nu sai khdc, ehi ed trinh tu DNA tuong ddng vdi gRNA-4 ed 1 Nu sai khde ndm trong viing loi (seed region) cua crRNA nen it ed kha nang xay ra dot biln sai so vdi gRNA-1.
Ten
gRNA-2 gRNA-4 gRNA-6 gRNA-10 gRNA-16
Bang 2. C^c trinh tir DNA gidng/gan giong Trinh tu doan DNA gidng/gan
gidng gRNA trong he gen liia ACGGCAAG [CAGGTCAGCAGC]
AATATTTA[CTCCCTCCAACC]
TGGTTAGT[ACCTGGGTGGAG]
GCGACGAT[GAGCTTAGCACC1 GGGCCACT[GGCGAAGGCCCA]
GGGCCACT[GGCGAAGGCCCA]
AGGGCGAC [GGCGAAGGCCCA]
AGGGCGAC [GGCGAAGGCCCA]
AGGGCGAC [GGCGAAGGCCCA]
AGGGCGAC[GGCGAAGGCCCA]
AGGGCGAC [GGCGAAGGCCCA]
AGGGCAGC[GGCGAAGGCCCA]
AGAGCGGC[GGCGAAGGCCCA|
AGAGCGGC[GGCGAAGGCCCA|
AGAGCGGC[GGCGAAGGCCCA|
Vitri nhan bidt
E
vdi gRNA trong h6 gen lua Khoang each den Exon gSn
nhat 986 5059 0 990 1909 2937 615 1089 1199 3093 5059 1192 137 578 4586
Ma sd gen dich
Os01g0624700 EP1OSAG00000012214 Osllg0140300 Os07g0298900 OsiOgoienoo EP1OSAG00000009160 EP1OSAG00000006462 Os02g0570500 EP1OSAG00000019798 EP1OSAG00000009599 EP1OSAG00000019536 EP1OSAG00000020018 EP1OSAG00000022991 EP1OSAG00000013860 EP1OSAG00000024561 Ghi chd' Chd tiong ngoac [] the hidn trinh tu
hien vi tri sai khic so vdi vitiiNu tuong ung tidn gRNA Nhu vay, tdng hpp edc kit qud phan tich du dodn cau tnic vd tinh dae hieu cd thi xac dinh eac crRNA ed t h i su dung cho nghien euu gdy dot biln ehinh sira promoter/gen OsSWEETMTBR theo ea ehi ghep ndi dilm eudi khong tuong ddng (Non- homologous end joining) bdng he thdng chinh sira gen CRISPR/Cas9 [4], hao gdm: gRNA-1 gay dot
vdi vimg Idi (12 nu) cda crRNA; chu in dam thd
biln TalC, gRNA-10 gay dot bien ddng thdi AvrXa7, PthXo3 vd Tal5, gRNA-16 gay bdt hoat hodn toan gen OsSWEETMTBR Kit qua eiia nghien euu ndy Id ca sd eho nghien ciiu sau ndy nhdm tao ra ddng liia TBR225 khdng benh bac la bang eong nghe ehinh sua gen.
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 12/2018 47
KHOA HOC CONG N G H £
4 . KET LUAN
Promoter OsSWEETMTBR lien quan din qua trinh xam nhilm cua vi khudn bac la Xoo tren gidng ilia TBR225 da duac phan lap va giai trinh tu ddy du.
Vung trinh tu da phan lap ddi 1392 bp, bao gdm doan promoter ddi 1340 bp va doan Exon I dai 52 bp, gidng 99% so vdi OsSWEETM cua gidng liia Japonica (Niponbare, ma sd AP014967.1) va 100% so voi OsSWEETM ciia gidng lua Indiea (Shuhui498, ma sd CP018167.1). Promoter OsSWEETMTBR chua h6p TATA dae tnmg vd 4 vi tri nhan bilt cua vi khuan Xoo, bao gdm TalC, Tal5, PthXaS va AvrXa7. Ba edu tnic gRNA da dugc thilt k l phuc vu nghien eiiu ehinh sua promoter/gen OsSWEETMTBR bdng cong nghe CRISPR/CAS9. Kit qud ciia nghien cuu dat n i n mdng cho dinh huong tang cuong khd nang khang benh bac la cua gidng lua chu luc TBR225 bdng cong nghe chinh siia he gen.
UncPMcm
Nghidn euu dupe hd tia Idnh phi tii Du in 'Nghidn edu ung dung cdng nghd ehi thi phan tu va ehi:: ^da he gen tiong chpn tao gidng liia nang suit, chat lui^pg, chdng chiu sau benh va bat Ipi ngoai cinh ", thuoe Chuong trinh Ddi mdi eong nghe qudc gia (Ma sd: Di/i.36.DN/18). Chung toi xin tran ti'ong cam on.
TAI UEU THAM KHAO
1. Antony G., Zhou ]. Huang S., Li T., Liu B., White P., Yang B. (2010J. Rice xal3 recessive resistance to bacterial blight is defeated by induction of the disease susceptibility gene OsllNS. Plant Cell, 22(ll):3864-76.
2. Blanvillam-Baufume S., Resehke M., Sole M., Auguy P., Doueoure H., Szurek B., Meynard D., Portefaix M., Cunnac S., Guiderdoni E., Boch J., Koebnik R. (2017). Targeted promoter editing for nee resistance to Xanthomonas oryzae pv. oryzae reveals differential activities for SWEETO-indncmg TAL effectors. PlantBiotechnol J., 15(3):30r-317.
3. Boch J., Bonas U. (2010). Xantiiomonas AvrBs3 family-type ill effectors: discovery and hinetion. Annu. Rev. Phytopatiiol, 48:419-36.
4. Bortesi L , Fischer R. (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv., 33(l):41-52.
5. Chen L. Q., Qu X. Q., Hou B. H.. Sosso D., Osorio S., Femie A R, Frommer W. B. (2012).
Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science, 335(6065):207-ll.
6. Doyle J. J. and Doyle J. L (1990). Isolation of plant DNA from fresh tissue. Focus, 12:13-15
7. Hutin M., Sabot F., Ghesquiere A , Koebnik R., Szurek B. (2015). A knowledge-based molecular screen uncovers a broad spectrum OsSWEETM resistance allele to bacterial blight from vnXd rice.
Plant J, 84(4):694-703.
8. Liang G., Zhang H., Lou D., Yu D. (2016).
Selection of highly efheient sgRNAs for CRISPR/Cas9-based plant genome editing. Sci Rep., 6:21451.
9. Nakashima K., Jan A., Todaka D., Maruyama K., Goto S., Shinozaki K., Yamaguehi-Shmozaki K.
Comparative functional analysis of six drought responsive promoters in transgenic rice. Planta, 239(l):47-60.
10. Pranamika S., Bora L. C , Puzari K. C, Baruah A. M., Baruah R., Talukdar K., Kataky L, Phukan A (2017). Review on Bacterial Bhght of Rice Caused by Xanthomonas oryzae pv. oryzae. Different Management Approaches and Role of Pseudomonas Buorescens As A Potential Biocontrol Agent. Int J.
Curr. Microbiol App. Sci, 6(3):982-1005
11. Sambrook J., Russel D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spnng Harbour Laboratory. Cold Spring Harbour, NY.
12. Smith L M., Sanders J. Z., Kaiser R J., Hughes P., Dodd C , Connell C. R, Hemer C , Kent S. B., Hood L. E. (1986). Fluorescence detection in automated DNA sequence analysis. Nature,
48 N O N G NGHIEP V A PHAT TRIEN N O N G THON - KY 1 - THANG 12/2018
KHOA HOC CONG NGHi
321(6071):674-679. close rice SWEET genes confer TAL effector- 13. Streubel ] . , Pesce C , Hutin M., Koebnik R., mediated suseeptihihty to Xanthomonas oryzae pv.
Boch J., Szurek B. (2013). Five phylogenetically oryzae NewPhytol.,2'Xl:«i&-i\.
ISOLATION AND DESIGN HIGH SPECIFICnY gRNAs FOR E D m i N G PROMOTER OsSWEEn4 IN ORDER TO IMPROVE BACTERIAL LEAF BUGHT DISEASE RESISTANCE
OFTBR225 RICE VARIETY
Phung Thi Thu HuongS Nguyen Duy PhuongS Pham Xuan Hoi^
^Agricultural Genetics Institute Summary
OsSW^£Tfamily encodes sugar transporters which are involved in the bactenal leaf blight (BLB) disease.
In this study, promoter OsSWEETt4v/as isolated from the total DNA of the Xanthomonas oryzae pv. oryzae (Xoo) susceptible TBR225 variety rice. The 1392-bp isolated DNA sequence contains four cis acting elements reconized specifically by Xoo TAL proteins, including TalC, Tab. PthXaS va AvrXa7. OsSWEETt4- 7B/e22;showed the similarity of 99% and 100% to OsSWEET14of GeneBank-registed Japonica (Niponbare.
AP014967.1) and Indica (Shuhui498, CP018167.1) vaneUes, respectively. Based on the isolated DNA sequence, three gRNAs were designed for modifying the TAL effector binding sites on the OsSWEin'14 promoter and knockouting the OsSWEITtt gene using CRISPR/CAS9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) technology m order to enhance the resistance of TBR225 nee veriety. This research is the basis for generating BLB disease resistant and high yielding rice varieties using genome editing technology in Vietnam
Keywords: Bacterial leaf blight CRISPR/CAS9, OsSWEETM, T4L eifector Xanthomonas oryzae
NgirW phan biSn: PGS.TS. La Tuan Nghia Ngay nhan bai: 21/9/2018
Ngay thong qua phan b i t o 22/10/2018 Ng4y duySt dang: 29/10/2018
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 12/2018 49
