苦荞愈伤遗传转化体系的优化及用于Ft CHS1的过表达分析  

Optimization of Callus Genetic Transformation System and Its Application in FtCHS1 Overexpression in Tartary Buckwheat

作　　者：赵海霞[1] 肖欣 董玘鑫 吴花拉 李成磊[1] 吴琦[1] ZHAO HaiXia;XIAO Xin;DONG QiXin;WU HuaLa;LI ChengLei;WU Qi(College of Life Science,Sichuan Agricultural University,Ya’an 625014,Sichuan)

机构地区：[1]四川农业大学生命科学学院,四川雅安625014

出　　处：《中国农业科学》2022年第9期1723-1734,I0002,I0003,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31871699)。

摘　　要：【目的】建立和优化苦荞愈伤组织遗传转化体系,为苦荞基因功能验证及分子育种提供研究工具。【方法】以苦荞品种“西荞二号”为材料,对苦荞愈伤遗传转化条件进行优化,包括苦荞外植体类型、诱导愈伤的激素比例、继代培养基的激素比例及农杆菌类型。利用苦荞类黄酮生物合成关键酶基因FtCHS1的过表达验证优化后的遗传转化体系。通过PCR筛选和荧光观察鉴定阳性株系,采用紫外分光光度法和高效液相色谱法(high performance liquid,HPLC)测定花青素及黄酮醇支路代谢物含量,使用实时荧光定量PCR分析类黄酮合成相关基因的表达,比较FtCHS1过表达愈伤组织与对照组的差异。【结果】苦荞诱导愈伤组织的最佳外植体为下胚轴,其最适诱导培养基为MS+0.8 mg·L-1 6-BA+3.5 mg·L-1 2,4-D,诱导率达72%;最优继代培养基为MS+3 mg·L-1 6-BA+1 mg·L-1 KT,愈伤组织增殖率与增殖系数分别为98%和1.09;转化过程中的最佳农杆菌是GV3101,转化效率达31.3%;FtCHS1过表达愈伤组织中,花青素、芦丁和杨梅素的含量显著高于对照(P<0.05),山奈酚和槲皮素的含量极显著高于对照组(P<0.01);外源FtCHS1的过表达对转基因愈伤组织中5个内源同源基因FtCHSs的表达水平没有影响(P>0.05),而FtCHI、FtF3H、FtFLS1、FtFLS2、FtFLS3和FtDFR1等黄酮合成途径关键酶基因均上调表达(P<0.05)。此外,特异性正调控黄酮醇合成的转录因子基因FtMYB5和FtMYB6上调表达,而花青素合成抑制子基因FtMYB8的表达降低(P<0.05)。【结论】建立了苦荞愈伤组织遗传转化体系,过表达FtCHS1的苦荞愈伤组织通过上调黄酮合成相关基因的表达增加类黄酮物质的积累。【Objective】To develop a novel tool for functional verification and molecular breeding in tartary buckwheat, this study focused on establishing and optimizing an efficient callus genetic transformation system. 【Method】Callus induction factors including different explants, ratios of diverse growth regulators, and Agrobacterium tumefaciens types were systematically evaluated using “Xiqiao No. 2” as the derived plant. We further overexpressed FtCHS1, a key enzyme gene involved in the biosynthesis of tartary buckwheat flavonoids in obtained calli to validate the optimized genetic callus transformation system. The positive transgenic lines were confirmed by PCR and fluorescent observation. Subsequently, the content of anthocyanins and metabolites in flavonol branch pathway were determined by UV spectrophotometry and High Performance Liquid Chromatography(HPLC), respectively.Furthermore, quantitative real-time PCR was performed to analyze expression levels of genes involved in flavonoid synthesis, in order to compare the differences between the FtCHS1-overexpressed calli and the control. 【Result】The optimal explant was hypocotyls and the optimal induction medium was the Murashige and Skoog(MS) medium supplemented with the addition of 0.8mg·L-1 6-BA(6-Benzylaminopurine) and 3.5 mg·L-1 2,4-D(2,4-Dichlorophenoxyacetic acid). The induction rate of calli grown on the above medium reached up to 72%. Moreover, the optimized subculture medium containing MS with the additives of 3 mg·L-16-BA and 1 mg·L-1 KT(Kinetin) increased the percentage and coefficient of callus proliferation to 98% and 1.09, respectively.Additionally, the best Agrobacterium tumefaciens in the transformation process was GV3101, and the transformation efficiency was up to 31.3%. The functional analysis of FtCHS1 overexpressing in transgenetic calli demonstrated that:(1) The accumulations of kaempferol and quercetin in transgenic calli overexpressing FtCHS1 were dramatically higher than those in control groups(P<0.01),and anthocyanin, rutin

关 键 词：苦荞 愈伤组织 遗传转化 查尔酮合酶基因 

分 类 号：S517[农业科学—作物学]