过表达TaPK-R1基因增强了小麦对纹枯病的抗性和耐冻性

TaPK-R1 Overexpressing Transgenic Wheat Lines Enhance Resistance to Sharp Eyespot and Freezing Stress

小麦纹枯病菌（Rhizoctonia cerealis）和倒春寒是小麦生产中重要的生物和非生物胁迫因子,本研究目的是利用转基因技术改良小麦的纹枯病抗性和耐冷性。利用构建的转基因载体pAHC25-MYC-Ta PK-R1,通过基因枪介导法,将小麦AGC蛋白激酶基因TaPK-R1导入春小麦品种扬麦20,获得TaPK-R1过表达的转基因小麦。对T1至T4代植株进行PCR、RT-PCR、qRT-PCR、Westernblot分析,筛选到3个转基因小麦株系,外源TaPK-R1基因能够在其植物体内遗传和超量转录,并翻译成蛋白质。利用致病力不同的R.cerealis菌株R0301和WK207,分别对3个转基因株系T_1至T_2代和T_3至T_4代进行纹枯病抗性鉴定,发现TaPK-R1过量表达提高了转基因小麦的纹枯病抗性,3个转基因株系各世代的纹枯病平均病级为1.16~2.11,病情指数为23.20~42.10,而对照扬麦20的纹枯病病级为2.55~3.60,病情指数为51.00~72.00。用-9℃低温处理三叶一心期小麦幼苗24 h,对照扬麦20的存活率为17%,3个转基因小麦株系的存活率分别为52%、79%和96%。上述结果表明,TaPK-R1过量表达能显著增强小麦对纹枯病的抗性及对冻害的耐受性,所获得的3个转基因小麦株系可作为潜在的抗源材料。

Sharp eyespot caused by Rhizoctonia cerealis is a soil-borne disease and freezing stress is one of the major abiotic stresses in wheat production. The object of this study was to improve wheat resistance to sharp eyespot and freezing using transgenic technique. The transformation vector p AHC25-MYC-Ta PK-R1 expressing the wheat AGC protein kinase gene Ta PK-R1 was constructed and transformed into the spring wheat Chinese cultivar Yangmai 20 through particle bombardment. The transformed T1 to T4 plants were subjected to PCR, RT-PCR, q RT-PCR, and Western blot analyses. Three transgenic wheat lines were generated and screened, in which the introduced Ta PK-R1 transgene was inherited and expressed in higher level. After inoculation with R. cerealis virulent-isolate R0301 or WK207, these three Ta PK-R1-overexpressing transgenic wheat lines displayed significant improved resistance to sharp eyespot. The infection types of these 3 transgenic lines in T1 to T4 generations were 1.16–2.11, and their disease indices were 23.20–42.10. At the same time, the infection types and disease indexes of the non-transformed wheat Yangmai 20 were 2.55–3.60 and 51.00–72.00, respectively. The three-leaf wheat seedlings were treated with ？9？C for 24 hours. Freezing tolerances of the three transgenic lines were dramatically improved, whose survival rates were 52%, 79%, and 96%, respectively, and significantly higher（P 0.01） than that of the non-transformed Yangmai 20（survival rate of 17%）. Our results indicate that resistance/tolerance to sharp eyespot and freezing stress could be significantly enhanced in Ta PK-R1 overexpressing transgenic wheat. The three transgenic lines may serve as potential resource in wheat breeding aiming at resistance improvement to sharp eyespot and freezing stress.