KHOA HOC C 6 N G N G H |
PHAIM LAP V A DAC TRLTIXIG CUA CHUIMG VI KHUAlV IVqi 5IIVH KiCH THICH SIIXIH TRUQIXIG THIJC VAT
TIJ CAY CA DAI
Hoang Hoa Long', Phara XuSn Hdi' T(!)MTAT
Cac loai cSy dai U ngudn cung cap vi k h u ^ n^i sinh phong phu c6 kha nSng kich thich sinh tniong ciia cSy chu. Vi khu& nOi sinh kich thich sinh tnidng cSy chi thfing qua sinh tdng hgp enzym 1- aminocyclopropane-l-cacboxylat (ACC) deaminaza g6p p h ^ lAm giam ham lugng etylen c6 hai trong cSy va san sinh hodc m6n lAA (indole-3- ax^tic axit) di^u h6a sinh truang ciia cSy. Trong nghifin cuu n^y, cac chung vi khu£n n^i sinh dupe phan l§p tir rk, thSn vA 1^ ciia Solanum nigrum, mOt loai c^ d?i. CAc d5c tinh nhir long hpp enzym ACC deaminaza, san sinh lAA, phSn huy photphat v6 CCT vi kha nSng tdn t^i trong cSy non la nhOng dac trung ccr ban li6n quan d^n kich thich sinh tnidng thi;c v§t ciia cic chung vi khuan niiy.
Ngoai ra, chiing cdn c6 kha nang Urn tang sire sdng cua cSy non. K^t quS cho th^y, hdu h^t cAc chung vi khudn tdng hgp ACC deaminaza va lAA d^u lam ting chi^u did rk theo quan h^ tuyffn tinh. Vj tri ton t^i ciia cac chiing vi k h u ^ nOi sinh b^n trong m6 cSy chii dupe xAc dmh th6ng qua bi^n ngp plasmit mang gien m3 hoa Green Fluorescent Protein (GFPuv) vko t6 bao vi khuln. T6m 1^, nghifin ciJru ndy da ph^t hi^n dupe cAc chiing vi k h u ^ npi sinh ti^m nang kich thich sinh truong cSy chCi vi ca ch^ Lfin quan.
TizlihAa: ACC deaminaza, ciy d^, indole-3-ax€tic axit, siitSnt$iva vi khu^ n^i sinh.
LBATVANDE
Vi khuan n6i sinh t6n tai b ^ trong mo thuc v§t va khong gay cac tri^u chiing benh cho cay. Ciing gidng nhu vi khuSn bi^u mo, vi khuan noi sinh dong vai tro quan trong trong sinh truong va phat trien ciia cay chii thong qua cac ca ch^ khac nhau, vi du nhu CO dinh N2 tir khi quyen cho cay; san sinh cac hooc m6n thuc v | t nhu indole-3-axetic axit (lA^, gibberellin, cytokinin, etylen..., sinh tong hop enzym l-aminocyclopropane-l<acboxylat (ACC) deaminaza lam giam luong etylen c6 hai trong cay va kha nSng san sinh mot so chat hiiu ca bay hoi (volatile organic compound) (Hardoimetal.,2008).
Cac loai cay dai dirpc xem la nguon cung c^p phong phii vi khuan noi sinh kich thich sinh truong thuc vfit do sv dong ti^n hoa va trai qua qud trinh chon loc tu nhi^n (M. Fredmann, unpublished). Mot so loai cay dai ciia liia, dau tuong, thuoc la, liia mach... dupe biet mang mot quan Ih^ vi khuan n6i sinh phong phii va c6 tac dung tich ctrc den su sinh truong va phat trien cua cSy chii. Ngoai ra, cic chiing vi k h u ^ noi sinh nay cung cd tac dung kich diich sinh truong cac loai CEiy trong khac nhu vi khuan nOi sinh phan l§p tir Nicotiana glauca, mdt loai thuoc la dai, CO kha nSng kich tiiich sinh tnrong cSy Arabidopsis (Domenech, 2007).
Do vay, khai thac ti^m nSng vi khuan npi sinh tir cay c^ dai Solanum nigrum de ling dung trong kich thich sinh truong cay chu da dupe ti^n hanh.
I . VAT U$IVA PHUONG PHAP
Vi k h u ^ noi sinh dupe phan lap tir r^, than va la ciia cSy ca dai (5, nignmi) moc ttr nhi&n o cac vung sinh thai khac nhau tai thanh pho Jena, C6ng hoa Lien bang Diic, theo phuong phap khii trung b^ mat mo (Surface disinfection) b4ng etanol 70% trong 2 phiit va hipoclorit natri 3% trong 5 phiit, sau do rtra lai b ^ g nuoc cat v6 triing 3 Ian. Nghien m6 da dirpc khii trimg, thu dich chiet, pha loang va cay tren m6i truong nuoi cay thich hpp (Long et al., 2008).
Cac dac di^m lien quan d^n kich thich sinh truong cay trong ciia vi khuan npi sinh nhu san sinh lAA c6 b6 sung hoSc khdng b6 sung tryptophan dupe xac dinh theo phuong phap ciia Brie (1991). T6ng hpp enzym ACC deaminaza sii diing ACC nhu mpt ngu6n N2 duy nh£(t dupe xic dinh theo Click va cpng su (1998). N6ng dd enzym dupe xac dinh b ^ g each do lupng alphaketobutyrat tao ra khi phan huy ACC.
Phuong phap phan hiiy phot phit v6 co sii dting Ca3(P04)2 nhu m6t ngu6n phot phat duy nhat trong moi truong Pikovskaya (PVIO nhu mo ta ciia Verma va cong stt (2001). Kha nang phan htiy ph6t phat dupe xac dinh khi vi khuan tao m6t viing trong suot xung quanh khuan lac tr^n mdi truong PVK 'Vien Di truyen Nong nghi?p
NONG NGHIf P VA PHAT TRIEN NONG THON - KY 1 - THANG 6/2012
KHOA HQC CONO NOHt
Phuong phap tai nhiJm vi khuSn nOi sinh vao cay ca d?i S. nigrum qua h?t da k h i triing bi mjt, dong th6i xac djnh anh huong ciia chiing 16n Stic s6ng cua cay non (seedUng vigor) theo phuong phap c i a Abdulbak va Anderson (1973). Siic s6ng cua cay non dugc xac djnh thdng qua cac chi tieu nhu; dO ndy mdm cua h^t va chiiu dai than rk. Xac djnh kha nang ton tai (colonization) trong m6 cay chii cua cac chiing vi khuin nOi sinh tiia hanh thdng qua bi^n nap gien phat h u ^ h quang green fluorescent protein (.GFPuv) nhu m6ta ciia Long vacOngsvt (2008).
•.KCrQuAviniAoiiiiN
1. £>ac tinh sinh hoc ciia vi k h u ^ nOi sinh li6n quan A4n kha nang kich Ifaich sinh trudng cay chii
Tdng s6 77 chung vi k h u & nOi sinh duoc phSn lap tir ri, than va la c i a cay ca d?i S. nigirnn mpc ti;
nhien b cac viing sinh thai khac nhau t?d thanh phd Jena - COng hda Lita bang Diic. Trong sd do 23 chiing c6 kha nang san sinh enzym ACC deaminaza tr6n mdi trudng tdi thidu c6 bd sung ACC nhu mpt nguAn N2 duy nhSt Mdt chiing cd kha nang san sinh lAA khi khdng bd sung tryptophan va 28 chiing tdng h()p lAA trong mdi trudng khi bd sung tryptophaa sau chiing vi k h u ^ c6 kha nang phan huy phdt phit vd CO va 24 chiing c6 kha nang tdn t^i trong md cua cay non (Bangl).
Bang 1. cac chiing vi khu& ndi sinh cd dac tinh sinh hoc Uta quan tin kich thich sinh tniAngc^dii Ngudn
gdc"
BGCRl BGCSLl BGCR2 BGCSL2 DR DSL DSR SSR Total
Tdngsd chiing
11 4 13 13 9 8 12 7 77
Sdchtin san sinh ACC
deaminazat 1 0 5 1 2 5 2 6 23
san sinh lAAii} K / ^ -Trp
0 0 0 0 1 0 0 0 1
+Tlp 3 1 5 2 5 5 2 5 28
f
Hda tan phdt phat VdC00 0 2 1 1 0 1 1 6
Tdn t^i trong cay non
2 0 5 2 3 4 2 6 24 ' Vi khu&i n0i sinh du^ phSn ]$p tu ri (R) vAtbinli (SL) tirc^c viing sinh thdi khAc nhau & thAnh pM Jena, C^ng hoa LB Buc;' ACC: 1-Aminocydopropan-l-cacboxylat (nmol alpha-ketobutyrate/mgprotein/gia);
*IAA:Indole^ax6ticaxit (pg/ml); Tip: DL-Tryptophan.
2. Anh hu6ng ciia lay nhi^m vi k h u & nOi sinh d^n sue s6ng ciia cSy non (seedUng vigor)
SUE sdng cua cay non S. nigrum (seedling vigor) dupe danh gia dtta tr^n anh huong ciia v i ^ lay nhiSm vi k h u ^ d^n d$ nay m ^ cua h^t, chi^u dai ciia than va xh cay non. L6 h^t ddi chiing dupe xu ly v6i ntr6c c^t v6 trimg. Trong sd 77 chiing vi khuSn ttiu n g h i ^ , 37 chiing lam tdng sue sdng ciia cay non. Trong sd 37 chiing nay, 22 chiing lam tang dO nay m ^ ciia hgt so v6i ddi chiing. Hai miroi bay chung (27) lam tang chi^u dai ciia rg va 11 chiing lam tang chi^u ddi ciia than so v6i ddi chiing. Mpt chiing lie eh^ sir nky m i n cua h^t va 4 chung lic chd sv ph6t tri^n cua rk h o ^ than cua cay non (Hinh 1).
3. Aoh hudng ciia lAA vd ACC deaminaza ciia vi k h u ^ d&i SI; phdt tri&i ciia h^ th cdy chii
Trong sd 16 chiing vi khuIn tong hpp ACC deaminaza, 6 chiing tdng hpp ACC deaminaza vdi
ndng do eao tir 200 d^n 700 nmol alpha- ketobutyrat/mg protein/gid da kich thich su phit tri^n ciia rk. DO dai ciia rh ti 1$ tuy^n tinh vdi nong dp enzym do vi k h u ^ ndi sinh sinh ra (Hinh 2A). Uy nhiSm h?it vdi cAc chung vi khudn nay da lam giam theo ti 1$ tuydn tinh vdi HAm lupng etylen trong cay (Hinh 2B). Mdt nhdm vi khuSn khac san sinh ACC deaminaza vdi ham lupng thf(p cung lam tang chi^u dai cua xh so vdi ddi chiing, chiing to cd co ch^ khac lifin quan d^n khS nang kich tiiich sinh trudng cSy chii cua cac chiing vi k h u ^ nay.
Ngoai ACC deaminaza, 14 chiing vi k h u ^ tong hpp lAA cd tac ddng d^n su phat tridn ciia xh cay non. Mpt sd chung vi khudn san sinh lAA b ndng do tir 1,1 ddn 11 pg/ml da lam tang chi^u dai ciia xk (Hinh 2C). Trong khi, c i c chiing vi khuSn tdng hgp lAA d ndng dO tit 93 ddn 115 pg/ml da lic ch^ su ph4t tridn cua xh.
N 6 N G NGHlgP VA PHAT T R I I N N 6 N G T H 6 N - irt' 1 - THANG 6/2012
KHOA HOC C O N G N G H |
Ngoai ra, khi xu ly hat tiiudc li vdi cic ndng do tu (Hinh 2D). Di^u ndy chiing td dnh hudng cua lAA khac nhau ciia lAA tong hpp cung cho kdt qua tuong l^n su phdt tridn ciia rk la phu thudc ndng dp.
0:0: a r t Chung vi khu^n
Hinh 1. Anh hudng ciia vi khuan noi sinh dSn do nay m4m ciia hat, chi6u dai than v^ r6 cua cay non (seedling vigor); miii t^n chi ra cac chiing vi khuan lam tang ty 16 nay mam cua hat hoac tang chieu dai ciia
than hoac r6.
Hinh 2. Quan h$ tuydn tinh gifla ndng dd enzym ACC deaminaza ciia vi k h u ^ vdi chidu dai rfi va sinh tdng hpp etylen trong cay (A vd B) vk quan h^ gifia ndng dd lAA cua vi khucb/IAA tdng hpp va su phat tridn
cua rh (C va D). Cac d vudng trong hinh ve bidu thj ede chiing vi k h u ^ san sinh ACC deaminaza va lAA vdi ndng dd k h ^ nhau.
NONG NGHlfP VA PHAT TRIEN N O N G THON - KY 1 - THANG 6/2012
4. Vj tri tdn tai ciia vi khudn ndi sinh trong md rfi cua cay chii
Dd xac dinh vi tri tdn tai eua cae chiing vi k h u ^ ndi sinh trong md cSy chu. MOt plasmit mang gien GFPuv dupe ehuydn \ko cdc chung vi khuSn n^y
giiip phat hi^n vj tri ciia chiing trong md cay chu dudi kinh hidn vi hu^^ih quang. Kdt qua tai phan lap cac chung vi k h u ^ nay cho thaiy chiing cd tiid tdn tJii trong md Ifin ddn 10^ cfu/g tuot d viing bidu bi va c k khoang gian bao (Hinh 3).
Chung BGCR2-8(1)GFP
Chung DR5GFP
Hinh 3. Si^ tdn tai cac chung vi k h u ^ bidn nap GFPuvtxoxi^ md rk quan sat dudi kinh hidn vi hu^nh quang nr.KEruiAN
-Trong sd 77 chiing vi k h u ^ npi sinh dupe phan lap va tuydn chpn tir rd, than va la cua cSy ca d ^ S nigrum d Jena, Cong hda lifin bang Diic cd 37 chung lam tSng siic sdng ciia cay non. Cac chung nay mang mot trong sd cac d§c tinh sinh hpc liSn quan ddn kich thich sinh trudng cay chii nhu tdng hpp ACC deaminaza, lAA, ph§n huy phdt phat vd ca va kha nang tdn tai trong md cay chii.
- Phan tich quan h& tuydn tinh giua ndng dd ACC deaminaza/IAA do vi khuan ndi sinh tdng hop va chidu dai rd cho thj(y kha nang kich thich sinh tntdng r^ cay ea d ^ cua cae chung vi k h u ^ ndi sinh n^y liSn quan chu ydu ddn tdng hpp lAA va enzym ACC deaminaza. Ngoai ra, kha nang ^ n tai cua chiing b6n trong md rd cay chii la nhan td quan trpng quyet dinh kich thich sinh trudng cua cay chu.
- Nghifin ciiu nay md ra tridn vpng img dung cic chung vi khu5n ndi sinh tir cac loai cay d^i dd kich thich sinh trudng cSy trdng. Ngoai ra, kidn nghi khai thae va bao tdn ngudn gien cd Ipi cho cay trong ciia vi sinh v^t ndi sinh tir cac loai cay dai d Vi^t Nam.
TAIUEUDIAMKHAO
1. Abdulbak A. A., Anderson J. D., 1973. Vigor determination in soybean seed by multiple criteria.
Crop Science 13:630-633.
2. Domenech J., Ramos S. B., Probanza A, Lucas G. J. A., Gutierrez M. R J., 2007. Elicitalion of systemic resistance and growth promotion of Arabidopsis thaliana by PGPRs from Nicotjaiia glauca: A study of the putative induction pathway- PlantSojI29Q: 43-50.
10 N O N G NGHI?P V A PHAT TRIEN N 6 N G THON - ICfr 1 - THANG 6/2012
KHOA HOC C O N G N G H |
3. GUck B. R., Penrose D. M., Li J. P., 1998. A growth in a species-specific manner; phytohormone model for the lowering of plant ethylene manipulations do not result in common growth concentrations by plant growth-promoting bacteria. / responses. PLoS ONE 3(7): e2702.
Theor. Biol. 190:63-68. doi:10.1371/joumal.pone.0002702.
4. Hardoim P. R., van Overbeek L S., van Elsas 6. Verma S. C , Ladha J. K., Tripathi A. K., 2001.
J. D., 2008. Properties of bacterial endophytes and Evaluation of plant growth promoting and their proposed role in plant growth. Trends colonization ability of endophytic diazotrophs from Mc7To6/oyi6: 463-471. deep water rice. Journal of Biotechnology ^l: 127-
5. Long. H. H.. Schmidt D. D., Baldwin I. T., ^^l- 2008. Native bacterial endophytes promote host
ISOLATION AND CHARACTERIZATION OF PLANT GROWTH PROMOTING BACTERIAL ENDOPHYTES FROM A WILD SOLANACEAE PLANT SPECIES
Hoang Hoa Long, PhamXuan Hoi Sununary
Wild plants harbor a diverse community of bacterial endophytes which can positively affect host plant growth. Changes in plant growth frequently reflect alterations in phytohormone homoeostasis by plant- growth-promoting (PGP) rhizobacteria which can decrease ethylene (ET) levels enzymatically by 1- aminocyclopropane-l-carboxylate (ACC) deaminase or produce indole-3-acetic acid (lAA). Bacterial endophytes were isolated from roots, stems and leaves of the field-grown Solanum nigrum plant, a native wild Solanaceae plant species; their PGP traits (ACC deaminase activity, lAA production, inorganic phosphate solubiLzation and seedling colonization) were characterized; and their effects on ttie seedling vigor were determined. A majority of isolates that promoted root growtti were associated with ACC deaminase activity and lAA production. Colonization of bacterial endophytes inside the root tissues was explored by transforming them with plasmid carrying the gene encoding green fluorescent protein {GFPuj^
and observing the inoculated tissues under fluorescence microscope. In short, the research opens a new trend in using native/natural bacterial endophytes with PGP traits from wild plant species to enhance yield of the cultivated crops.
Keywords: ACC deaminase, bacterial endophyte, colonization, indole-3-acetic acid, wild plant
Ngudi phan bi^n: GS.TS. Nguydn Quang Thach Ngay nh4n bai: 20/2/2012
Ngay thdng qua phan bi^n: 21/3/2012 Ngay duy^t dSng: 3/4/2012
, NdNG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 6/2012
