沙糖橘晚熟突变体果实中高水平游离IAA积累机制研究

前期研究发现沙糖橘晚熟突变粤农晚橘和已知由脱落酸和乙烯相关突变无关，而与高水平游离IAA相关，这是柑橘中从未报道过的晚熟机制。RNA seq研究发现突变体和对照果实中IAA合成和降解途径基因表达无差异，而编码催化结合态IAA生成的CsGH3.06蛋白基因表达量显著低于对照。本研究将采用基于DIA的蛋白质组学方法分析突变体和对照中IAA代谢途径的差异表达蛋白；测定两种果实中催化IAA生成限速酶YUC和降解关键酶IAAO和POD活性，明确酶活是否存在差异；研究突变体和对照果实的总蛋白提取物和CsGH3.06重组蛋白催化IAA-氨基酸结合反应速率的差异，分析CsGH3.06蛋白活性差异在游离IAA积累中的作用；未成熟沙糖橘果实中VIGS沉默CsGH3.06基因和草莓中过表达CsGH3.06基因，研究CsGH3.06在果实IAA积累中的作用。综合解析粤农晚橘果实中积累高水平游IAA的机制。

Previous studies showed that the late maturing phenotype of Yuenongwanju, a mutant of Shatangju (Citrus reticulate), is caused by mutations in neither ABA nor ethylene pathways, but caused by a previously unreported mechanism that results in a significantly higher level of free IAA in immature fruits. Results of RNA-seq showed that there was no significant differences in the expression of IAA synthesizing and degrading genes except that the CsGH3.06 gene, which encodes the key enzyme converting free IAA to IAA-conjugates, was significantly down-regulated in the fruits of the mutant as compared with that in normal shatanju fruits. In this proposed study, the mechanisms causing accumulations of free IAA will be thoroughly analyzed. Differentially expressed proteins involved in IAA metabolism pathways in the fruits between Yuenongwanju and control will be compared and identified by using the data-independent acquisition (DIA) proteomics. The activities of YUC, the rate-limiting factor in the auxin-biosynthetic pathway, and IAAO and POD, the two key enzymes for the degradation of IAA, will be determined in both the mutant and the control fruits to clarify if there would be a reduction in their activities. The activities of CsGH3.06 will be assayed for total protein extracts and for CsGH3.06 recombinant protein, to determine if there would be a difference between the enzymatic activities of both the mutant and the control. CsGH3.06 gene will be silenced by using VIGS in the immature green fruits, and will be over-expressed by using agrobacterium-mediated genetic transformation in strawberry to unravel the functions of the gene on accumulation of free IAA. Results of the proposed research should be enough to allow us to understand the mechanism underlying the high level free IAA in the green fruits of Yuenongwanju.