油菜糖基转移酶BnIRX14基因家族鉴定及遗传转化

Identification of BnIRX14 Gene Family and Genetic Transformation in Brassica napus

糖基转移酶在植物抗逆和发育调控中发挥着重要作用,为发掘糖基转移酶BnIRX14基因家族成员,解析在甘蓝型油菜中的生物学功能,该研究利用前期在甘蓝型油菜中克隆到的BnIRX14基因,采用序列比对和遗传转化的方法,进行BnIRX14基因家族成员鉴定和功能验证,以探讨BnIRX14基因家族在油菜发育中调控机理,为油菜杂交育种和抗逆育种提供理论依据。结果表明:(1)经基因组数据库比对分析在甘蓝型油菜中成功鉴定到3个糖基转移酶不同亚家族的11个BnIRX14家族成员,它们均具有糖基转移酶GT43家族成员结构域特征,其中有8个基因分别被定位在6条不同染色体上,3个亚家族在基因结构和保守元件中具有较大特异性。(2)利用农杆菌介导转化法,获得BnIRX14基因RNA干扰转基因油菜株系20株,经PCR检测,确定5株阳性转化体。(3)表型鉴定发现,有2株阳性转化株的花柱头至花柱中央为一孔状空腔,子房较野生型明显膨大,且柱头表面授粉后不能结实,表现雌性不育;其他3个阳性株花器结构发育正常,但植株茎、枝表皮有液体渗出,呈露珠状粘附在茎、枝表面。(4)实时荧光定量PCR分析显示,转BnIRX14基因油菜阳性植株花、角果、叶中的BnIRX14基因表达量均显著低于野生型,且9#阳性不育株的角果中BnIRX14基因表达低于其他转基因干扰株系,说明BnIRX14基因在干扰转化阳性植株中的表达受到显著抑制。研究推测,BnIRX14基因可能参与油菜雌蕊的发育和次生物质代谢。

Glucosyltransferase plays an important role in plant stress resistance and development regulation.In order to explore the members of the glucosyltransferase BnIRX14 gene family and analyze its biological function in Brassica napus,the study based on BnIRX14,which was cloned from B.napus and adopted the methods of sequence comparison and genetic transformation,we identified BnIRX14 gene family members and conducted functional verification to explore the regulation mechanism of BnIRX14 gene family in B.napus development,and to provide theoretical basis for cross breeding and resistance breeding in B.napus.The results showed that:(1)11 BnIRX14 family members belonging to 3 different subfamilies of glucosyltransferase were successfully identified in B.napus by genomic database comparison analysis.All of them had the domain characteristics of glucosyltransferase GT43 family members,among which 8 genes were located on 6 different chromosomes.3 subfamilies showed great specificity in gene structure and conserved elements.(2)Twenty transgenic B.napus lines with BnIRX14 RNA interference were obtained by Agrobacterium-mediated transformation,and 5 positive transformation lines were identified by PCR.(3)Phenotypic identification showed that there was a hole-like cavity from the top to the center of the stigma of the two positive transformation lines,the ovary was significantly enlarged than that of the wild type,and the stigma was not fertile after pollination,indicating female sterility.The other three positive plants had normal flower structure,but the stem and branch of the plants had liquid exudation,which adhered to the surface of the stem and branch in the shape of dew.(4)Real-time quantitative PCR(RT-qPCR)analysis showed that the BnIRX14 gene expression in the flowers,kernels and leaves of B.napus transgenic positive plants was significantly lower than that of the wild type,and the BnIRX14 gene expression in the kernels of the 9#positive sterile plant was lower than that of other transgenic interference lines.The results showed that BnIRX14 gene expression was significantly inhibited in the interference-transformed positive plants.It is speculated that BnIRX14 gene may be involved in pistil development and metabolism of secondary substances in rape.