紫花苜蓿细胞壁果胶解聚调控及其缓解苜蓿铝毒害的机制研究

Alfalfa is one of most valuable grass species for livestock, but its growth is greatly restricted by aluminum toxicity in south of china due to much low pH in soil, which strongly restricts the fresh alfalfa supply to develop low rate of food consume for raising livestock such as pigs, ducks and geese. Thus, it is important to study the Al toxicity in alfalfa and breed new alfalfa varieties with high Al tolerance. Both of polygalacturonase (PG) and IAA have the functions of regulating Al absorption in cell wall, There is a close relationship between the PG and IAA on regulating Al absorption and transport in cell wall, but its mechanism is unclear. The project will focus on studying the effects and mechanisms of PG and IAA on Al absorption and transport by the ways of transgenosis and changing IAA content in alfalfa seedling. The main research contents are as follows: 1) the function of MsPG1 and MsPG2 genes on the tolerance of Al toxicity in alfalfa; 2) the effects of MsPG1 and MsPG2 genes on the accumulation and transport of Al in cell wall and on the cell structure stability of Al stressed alfalfa root tips; 3) the regulation of MsPG1 and MsPG2 gene expression; 4) the effects of IAA on gene expression, protein accumulation and enzyme activity of MsPG1 and MsPG2 in Al stressed alfalfa seedling; 5) the effects of IAA and MsPGs genes on Al absorption of cell wall, cell structure and physiological response process of cell wall.

紫花苜蓿是畜禽养殖最有价值的牧草之一。然而，南方红壤土普遍呈强酸、高活性铝（Al3+）的特性，严重抑制了苜蓿生长和高效种植，制约了新鲜苜蓿的供应及其对猪、鹅等发展节粮养殖的支撑作用，因此，开展紫花苜蓿耐铝毒研究意义重大。多聚半乳糖醛酸酶（PG）与IAA在调控细胞壁铝吸附和转运过程中存在密切关系，但机理尚不清楚。本课题采用转基因和增减幼苗IAA含量相结合的方法，研究PG与IAA对苜蓿耐铝毒能力的调控作用和机制，重点研究：1）紫花苜蓿MsPG1和MsPG2(统称MsPGs)基因的耐铝毒功能；2）MsPGs基因对苜蓿细胞壁的果胶特性、铝吸附性能和细胞结构稳定性的影响；3）MsPGs基因的表达调控分析；4）IAA信号对铝胁迫苜蓿MsPGs在基因、蛋白和酶活性水平的调控作用；5）IAA与MsPGs基因对细胞壁果胶特性、铝吸附性能、细胞结构和细胞壁生理特性的影响，为培育铝毒耐受型紫花苜蓿奠定基础。

细胞壁是植物根系富集Al的主要部位，具有降低进入细胞内Al3+数量，保护共质体和细胞器免受铝毒害的功能。多聚半乳糖醛酸酶（polygalacturonase，PGs）是植物体主要的果胶解聚酶，影响果胶的结构和功能，但其影响植物抗铝毒性能的研究尚属空白。本课题围绕2个紫花苜蓿果胶解聚酶基因MsPG1和MsPG4（MsPGs）调控苜蓿响应铝胁迫的作用和机制开展研究，通过获得MsPGs过表达和干扰表达转基因紫花苜蓿植株，证实了1）MsPG1和MsPG4（根据2020年最新公布的紫花苜蓿基因组数据，重新命名MsPG2基因为MsPG4）具有显著缓解紫花苜蓿铝毒害的功能；2）MsPGs主要水解细胞壁碱溶型果胶（ASP）和水溶型果胶（WSP），从而，降低铝胁迫下细胞壁果胶的聚集程度，增加细胞壁孔隙度和延展性，减少细胞壁果胶及其Al含量；3）MsNAC73直接结合MsPG4启动子，负调控其表达；4）IAA可增强H+-ATPase活性，促进细胞内H+向质外体空间分泌，降低细胞壁果胶负电荷功能团数量，减少果胶的Al吸附能力，从而，降低细胞壁Al含量，增强苜蓿的抗铝胁迫能力，本研究结果对培育铝毒耐受型紫花苜蓿新品种具有重要的理论价值和实践意义。