Screening of Wheat Genotypes Sensitive to Agrobacterium tumefaciens Infection and Transformation-文献传递-植物通论文库

摘要：利用C58c1农杆菌菌株（其携带的pPTN290质粒载体上含有nptⅡ 基因和GUS基因）为供体材料和小麦幼胚为受体材料，对我国35个春小麦基因型进行了农杆菌敏感性研究和转化研究。组织化学染色结果表明，PM97034、P187、巴140105、扬麦158、新春9号和克丰6号等基因型农杆菌感染后GUS基因瞬时表达率达到了15.0% ~ 30.0%，显著高于模式基因型Bobwhite。其余幼胚分别在含10-25mg/L G418培养基上诱导愈伤组织和分化植株，抗性再生植株经ELISA检测、PCR检测、叶片离体退绿检测、X-Gluc染色检测和Southerm blot检测，最终从新春9号和PM97034的农秆菌转化效率高于Bobwhite. Southerm blot检测结果同时表明，外源基因在多数转基因植株中表现为单拷贝整合。T₁代植株ELISA检测结果和PCR检测结果表明，外源基因在个别株系中的分离符合孟德尔遗传规律，而在多数株系中的分离偏离了孟德尔遗传规律。阳性植株与阴性植株分离比经X²。适合性检测，表明外源基因在多数株系中的遗传符合3：1分离规律。本文首次对不同小麦基因型进行了农秆菌敏感和转化效率研究，筛选到新春9号和PM97034两个对农杆菌比较敏感且转化效率比较高的基因型，可望广泛用于小麦转基因研究。

Abstract:Thirty-five Chinese spring wheat genotypes were tested for their sensitivity to C58c1 Agrobacterium strain’s (harboring a binary vector pPTN290 with nptⅡ gene and GUS gene) infection and final transformation. Six cultivars including PM97034, P187, Ba1401051, Yangmai 158, Xinchun 9 and Kefeng 6 were proved to be more sensitive than Bobwhite to the Agro-infection according to X-Gluc staining, with GUS gene transient expression frequency of 15.0% to 30.0%. Regeneration plants obtained from selection medium were identified by ELISA, PCR, leaf bleach and X-Gluc, and stable transformation events were obtained from Xinchun 9, PM97034 and Bobwhite by 3.3%, 2.0% and 1.1%, respectively. Southern blot analysis showed the alien genes integrated in most transgenic plants just by single copy. The alien genes in most T₁ transgenic lines segregated by Mendelian regulation, but the segregation ratio in a few T₁ transgenic lines was a little bit away from the expecting value, according to X²_c test. Xinchun 9 and PM97034 are suggested to be used in wheat Agrobacterium mediated transformation to replace Bobwhite for better result.

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叶兴国等：农杆菌敏感小麦基因型的筛选及其转化图版Ⅰ
YE Xing-Guo et al.: Screening of Wheat Genotypes Sensitive to Agrobacterium tumefaciens
Infection and Transformation PlateⅠ

A B
D E
C

W Pa Pa W Pa Pa
P
G H
K
CK CK CK

Explanation of PlateⅠ
Test of transgenic wheat pants by histochemic l staining, leaf bleach and ELISA for GUS g ne and nptⅡ gene respectively.
A: GUS gene transient expression in the immat
GUS gene stable expression in the leaves of t
expression in the anthers of transgenic plants; F
with nptⅡ gene (W: water; Pa: 300 mg/L paro
control and negative control pointed by arrow, re

aure embryos after cocultivation; B: GUS gene
ransgenic plants; D: GUS gene stable express
: GUS gene stable expression in the mature see
momycin solution; CK: negative control); H:
spectively.). etransient expression in the callus of 7 d after cocultivation; C:
ion in the spikelets of transgenic plants; E: GUS gene stable
ds of transgenic plants; G: leaf bleach test of transgenic plants
ELISA test of the transgenic plants with nptⅡ gene (positive
1CF

Screening of Wheat Genotypes Sensitive to Agrobacterium tumefaciens Infection and Transformation

农杆菌敏感小麦基因型的筛选及其转化

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