海岛棉GbHCT13基因的功能验证

Functional Validation of GbHCT13 Gene in Gossypium barbadense

该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉GbHCT13基因(GenBank登录号MW048849)在纤维发育中的功能。结果显示:(1)成功构建重组载体pCAMBIA3301-GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转GbHCT13基因拟南芥T3代植株4株;qRT-PCR检测表明,转基因植株中GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中CAD、CCoAOMT、PAL和4CL与GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转GbHCT13基因棉花植株3株;转GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的CAD、CCoAOMT、CCR、PAL 4个基因的表达均呈降低趋势,说明抑制GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明,GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与GbHCT13基因在模式植物拟南芥中的基本一致。

This study used Gossypium barbadense Xinhai 21 and Gossypium hirsutum ND203 as well as model plant Arabidopsis thaliana to explore the function of Gossypium barbadense GbHCT13 gene(GenBank accession number MW048849)in fiber development through transgenic and fluorescence quantitative detection methods.The results showed as follows:(1)the recombinant vector pCAMBIA3301-GbHCT13 was successfully constructed,and 4 Arabidopsis T3 transgenic plants with GbHCT13 gene were identified by Agrobacterium mediated transformation,herbicide resistance gene screening,and fluorescence quantitative detection.QRT-PCR analysis showed that the expression of GbHCT13 gene in transgenic plants was significantly increased compared with that of wild type.(2)The transgenic Arabidopsis thaliana overexpression of GbHCT13 made the plant grow more vigorously than the wild type at the same period,and the plant shape,leaf number,moss number and stem surface fur number were different from the wild type.Histochemical analysis showed that compared with wild-type stem,the primary xylem of transgenic Arabidopsis thaliana grew more active,the ducts were thickened,and the cross section area of ductal cell wall in secondary xylem was larger,but the medulla cells had no significant changes.Overexpression of GbHCT13 changed lignin synthesis pathway genes in Arabidopsis to different extent,and CAD,CCoAOMT,PAL and 4CL were positively correlated with the expression of GbHCT13.(3)Through field screening and molecular identification,3 transgenic cotton plants with GbHCT13 gene were successfully obtained.The cotton fiber elongation and fiber strength of GbHCT13 transgenic cotton increased.The silencing of GbHCT13 gene reduced the lignin content of cotton plants,the number of stem surface fur,the number of xylem vessel cells and the lignin deposition in the vessel cell wall,but the cotton plants did not have obvious dwarf phenomenon on the plant height.Moreover,the expression of CAD,CCoAOMT,CCR and PAL 4 genes in lignin synthesis pathway all showed a decreasing trend,indicating that the inhibition of GbHCT13 impeded the growth and metabolism of cotton and affected the initiation of fiber development.The results showed that GbHCT13 can affect lignin synthesis in cotton plants and regulate fiber growth and development,and its function is basically the same as that of GbHCT13 in model plant Arabidopsis thaliana.