植物防卫基因PvPGIP2和TaLTP4的克隆及其对小麦的转化

CLONING OF PLANT DEFENSE GENES PvPGIP2 AND TaLTP4 AND THEIR TRANSFORMATION TO WHEAT

多聚半乳糖酸醛酶抑制蛋白和脂质转移酶蛋白是植物产生的防卫蛋白,对植物病原真菌具有防御作用。本研究利用同源基因克隆策略和RT-PCR技术,成功克隆了1个菜豆的多聚半乳糖酸醛酶抑制蛋白(PvPGIP2)编码基因和1个小麦的脂质转移蛋白(TaLTP4)编码基因;由于它们在功能上具有协同作用,我们以pAHC25为骨架构建了PvPGIP2和TaLTP4基因双价表达载体,对该双价表达载体进行菌落PCR筛选、限制酶消化鉴定和测序分析,结果表明构建的PvPGIP2和TaLTP4双价基因表达载体是成功的,该载体包含PvPGIP2表达盒和TaLTP4表达盒,分别受玉米泛素基因(Ubiqiutin)启动子的驱动,选用来源于Ti质粒中胭脂碱合成酶基因的终止序列作为终止子(Tnos)。采用基因枪法轰击小麦品种扬麦18成熟胚愈伤组织2000枚,经过2次Bialaphos筛选,最终获得扬麦18再生植株112株,利用表达载体基因的特异引物对上述成活的转化植株进行PCR检测,获得PvPGIP2和TaLTP4均为阳性的植株12株,转化率为0.6%。

Polygalacturcuase-inhibing protein（PGIP） and Lipid-transfer protein（LTP） were defense proteins produced in plant,which inhibit the growth of pathogenic fungi.In this study,a PGIP encoding gene in Phaseouls vulgaris（PvPGIP2） and a LTP encoding gene in Triticum aestivum（TaLTP4） were successfully cloned by using homologous gene cloning strategy and RT-PCR technique.By using pAHC25 as a frame vector,we constructed PvPGIP2and TaLTP4binary expression vector.Then the binary expression vector was through preliminary screening by colony PCR and restriction enzyme digest identification and sequencing analysis.The sequencing result showed that the PvPGIP2and TaLTP4binary expression vector was successfully constructed.In this transformation vector,the PvPGIP2and TaLTP4genes were respectively driven by the maize ubiquitin（Ubi） promoter and terminated by the 3′non-transcribed region of the Agrobacterium tumefaciensnopaline synthase gene（Tnos）.Through microprojectile-mediated bombardment,the binary expression vector was transformed to 2,000 mature callus of wheat variety Yangmai 18.After twice rounds of Bialaphos screening 112 regeneration plants were finally obtained.The living transformed plants were subjected to PCR detection using primers specific to expression vector gene to conduct,12 positive plants with PvPGIP2and TaLTP4were obtained,and the conversion rate was 0.6%.