结论:构建了一个稳定的农杆菌介导的转化体系,BjuLKP2过度表达,导致植物变黄。为了验证植物中的基因功能,需要稳定有效的转化系统。建立农杆菌介导的芥菜芽孢杆菌转化体系,各种因素,包括外植体类型,激素组合和浓度,感染时间和浓度,进行了优化。最终,建立了可靠的系统,和两个BjuLKP2过表达(OE)系,表现出子叶变黄,射击技巧,叶子和花蕾,以及总叶绿素含量的减少,产生了。qRT-PCR分析显示,在BjuLKP2OE品系中,五个叶绿素合成基因显着上调,一个基因下调。此外,抗氧化能力测定显示APX活性降低,CAT和SOD,而BjuLKP2OE26中的POD活性增加。此外,叶绿素荧光诱导的动力学测定表明BjuLKP2OE26的光合能力降低。GUS分析显示BjuLKP2在各种组织中的表达,包括根,下胚轴,子叶,叶脉管系统,毛状体,萼片,花瓣,长丝,花柱和柱头基部,但不是种子。扫描电子显示海绵和栅栏组织中叶绿体超微结构的变化。总的来说,这些发现表明BjuLKP2通过降低叶绿素含量和改变叶绿体结构在植物黄化中起作用。
CONCLUSIONS: A stable Agrobacterium-mediated transformation system was constructed for B. juncea, and BjuLKP2 was overexpressed, leading to plant yellowing. A stable and efficient transformation system is necessary to verify gene functions in plants. To establish an Agrobacterium-mediated transformation system for B. juncea, various factors, including the explant types, hormone combination and concentration, infection time and concentration, were optimized. Eventually, a reliable system was established, and two BjuLKP2 overexpression (OE) lines, which displayed yellowing of cotyledons, shoot tips, leaves and flower buds, as well as a decrease in total chlorophyll content, were generated. qRT-PCR assays revealed significant upregulation of five chlorophyll synthesis genes and downregulation of one gene in the BjuLKP2 OE line. Furthermore, antioxidant capacity assays revealed reduced activities of APX, CAT and SOD, while POD activity increased in the BjuLKP2 OE26. Additionally, the kinetic determination of chlorophyll fluorescence induction suggested a decrease in the photosynthetic ability of BjuLKP2 OE26. GUS assays revealed the expression of BjuLKP2 in various tissues, including the roots, hypocotyls, cotyledons, leaf vasculature, trichomes, sepals, petals, filaments, styles and stigma bases, but not in seeds. Scanning electron revealed alterations in chloroplast ultrastructure in both the sponge and palisade tissue. Collectively, these findings indicate that BjuLKP2 plays a role in plant yellowing through a reduction in chlorophyll content and changes in chloroplasts structure.