BpMYB4基因在白桦中的遗传转化及低温胁迫应答反应

Functional study of BpMYB4 in birch response to low temperature stress

【目的】为了研究Bp MYB4基因在冷胁迫反应中的功能,对其进行了白桦转化试验。【方法】采用定量PCR技术对Bp MYB4基因在白桦的根、茎、叶、木质部、芽、茎段等18个组织部位的表达模式进行分析,然后克隆构建PGWB2-Bp MYB4植物过表达载体,通过农杆菌介导法进行白桦合子胚的遗传转化,采用PCR和荧光定量PCR技术检测转基因白桦株系中Bp MYB4基因的相对表达量,最后采用分光光度计法对Bp MYB4转基因白桦进行了8项生理指标的测定。【结果】相对于芽的表达量,Bp MYB4基因在第3片叶的叶脉和第1片叶与第2片叶之间嫩茎的表达量最高;白桦的遗传转化总共获得了13个过表达株系,且Bp MYB4基因已经成功整合到白桦基因组上,定量结果发现MYB-8、MYB-11、MYB-12转基因白桦中Bp MYB4基因的表达量相对于非转基因对照株系(NT)上调幅度最大;在低温胁迫下,过量表达Bp MYB4基因提高了白桦的超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性、花青素(OPC)含量、过氧化氢酶活性(CAT)、可溶性糖含量、可溶性蛋白含量和脯氨酸(PRO)含量,同时降低了转基因白桦的丙二醛(MDA)的含量。【结论】Bp MYB4转基因白桦在低温胁迫下能够产生具有保护作用的物质,具有一定的抵抗低温的能力,因此,可以将其培育成抗冻优选株系。

【Objective】Our objective was to understand the functions of the Bp MYB4 gene during low-temperature stress.【Method】We performed the following experiments in this study. Firstly,we analyzed the expression pattern of the Bp MYB4 gene in different tissues of birch,including roots,stems,leaves,xylem and buds. Secondly,we constructed the plant overexpression vector of PGWB2-Bp MYB4,and generated birch transgenic lines that overexpressed Bp MYB4 using Agrobacterium-mediated transformation. Then,a polymerase chain reaction( PCR) and quantitative real-time PCR( qRT-PCR) were used to identify the transformants. Finally,we examined eight physiological indexes of transgenic birch under low temperature using spectrophotometry.【Result】Campare with the expression of Bp MYB4 in buds,the highest expression level of Bp MYB4 was in the veins of the third leaf and the tender stems between the first leaf and the second leaf. We obtained 13 independent 35 S ∶ ∶ Bp MYB4 transgenic lines and PCR results provided preliminary evidence that exogenous Bp MYB4 was integrated into the genome of birch. qRT-PCR results indicated that Bp MYB4 expression in MYB-8,MYB-11 and MYB-12 lines was significantly higher than that in the non-transgenic birch( NT). Under cool stress,overexpression of the Bp MYB4 gene in birch increased enzyme activities of SOD,POD and CAT. Overexpression of Bp MYB4 also increased contents of OPC,soluble sugar,soluble protein,and proline. Compared to NT,the content of malondialdehyde( MDA) in transgenic birch was reduced.【Conclusion】The results indicated that Bp MYB4 transgenic birch could produce protective substances under cold stress,and it will have an ability to resist cold. It can be developed into a freeze-resistant preferred strain.