二色补血草盐腺发育MYB家族关键基因LBMYB1和LBMYB2功能研究

Salt glands are the only visible morphological structure that distinguishes recretohalophytes from all of non-halophytes and other halophytes. The salt gland plays a critical role in survival of recretohalophytes in high salinity environment by directly secreting excess salt onto the surface of its leaves and stems to avoid salt stress. In recent years, transcript factors such as MYB, bHLH and C2H2 type zinc finger proteins play a pivotal role in the regulation of epidermal cell fate determination such as trichome and stomata. Limonium bicolor Kuntze (Plumbaginaceae) is a typical exo-recretohalophyte that has salt glands but without trichome. The leaf transcriptomes at different stages of salt gland development showed that some transcript factors genes distinguishingly high expressed at the initiation and early development of salt glands. In this project, leaves of M2 mutants with abnormal salt gland including no salt gland, much less salt gland and much more salt gland compared with wild tpye and isolated bicelluar salt gland with digestion enzymes will be used to further screen differentially expressed transcript factor genes by high throughput RNA sequence. 1-2 MYB family key transcript factor genes controlling salt gland iniation and development will be screened by comparative analysis and annotation of transcriptomes of leaves at different stages of salt gland development, the mutants and isolated salt glands with four cells. The function of the key genes controlling salt gland development will be further identified by bioinformatics ananlysis and molecular techniques such as transgenic lines, in situ hybridization, Real-time PCR, and examination of salt gland density, strcucture and distribution of over-expression or silence lines, mutant complement and interaction ptoteins analysis of these transcript factors. Finally, the key transcript factor genes, functions and regulatory pathway involved in salt gland differentiation will be drawn.

盐腺是泌盐盐生植物区别于其他盐生植物和所有非盐生植物唯一可见的形态结构，将体内过多的盐分泌到体外，使植物能够在高盐环境中生存。大量研究表明，转录因子在控制叶表皮原细胞分化成腺毛和气孔过程中起关键作用。二色补血草为典型的泌盐盐生植物，有盐腺而无腺毛，盐腺不同发育阶段叶片转录组数据库分析发现某些转录因子基因在盐腺起始和发育初期特异性高表达。本项目将对盐腺发育突变体[无盐腺、盐腺发育停止在某一阶段、盐腺成簇(cluster)发育]和酶解分离获得四细胞盐腺复合体和同时期表皮细胞进行转录组测序，通过叶片、突变体和盐腺复合体三个层次转录组数据库比对注释，确保筛选到参与盐腺启动及发育的1-2个MYB家族关键转录因子。通过生物信息学分析、基因时空表达分析、转基因株系(过表达株系、基因沉默株系及突变体回补株系)盐腺密度、结构、分布等观察分析和这些转录因子可能互作蛋白分析，揭示这些转录因子控制盐腺发育的机制。

盐腺是泌盐盐生植物区别于其他盐生植物和所有非盐生植物唯一可见的形态结构，将体内过多的盐分泌到体外，避免盐害。然而，关于盐腺发育的分子机理尚不清楚。本项目以具有典型盐腺结构的泌盐盐生植物二色补血草为研究材料，主要研究LbMYBs在盐腺发育中的功能，相关结果在The Plant Cell, Frontiers in Plant Science 等 SCI刊物发表论文22篇。主要研究结果如下：（1）通过EMS化学诱变获得二色补血草盐腺发育突变体lb23-3，具有4-8组分泌细胞、毗邻细胞、内杯状细胞和外杯状细胞组成的盐腺，即有16、20、24、28和32细胞盐腺；而野生型盐腺4组分泌细胞、毗邻细胞、内杯状细胞和外杯状细胞组成的盐腺，即16细胞盐腺。这些突变体材料为进一步研究盐腺发育分子机制奠定了基础；（2）通过不同发育阶段叶片转录组数据，获得盐腺发育时期高表达的4个MYB家族关键转录因子(LbTRY、LbMYB5、MYB106、MYB23)，在全长克隆和生物信息学分析的基础上，利用CRISPR-cas9在二色补血草中进行敲除，发现盐腺出现组成细胞数减少的现象，说明这两个基因参与调控盐腺的发育，但不是关键基因；（3）在拟南芥中找到C2H2转录因子LbZP1的同源基因AtZP1，并通过CHIP和EMSA进行功能验证，发现AtZP1负向调控根毛发育的ZP1/RSL模型；（4）通过本项目研究我们完成了二色补血草基因组测序、初步建立了二色补血草EMS突变体库、构建叶片发育早期的酵母文库，特别是发现了调控盐腺发育的关键基因如LbTRY和LbMYB5，为继续筛选到控制盐腺发育的上游调控因子，揭示盐腺发育分子机制并最终利用转基因技术培育耐盐植物奠定基础。