番荔枝β-淀粉酶基因AaBAM3在果实淀粉降解中响应低温的转录调控机制

Starch is the main carbohydrate in Atemoya fruit. Our previous studies found that starch degradation was directly related to the softening and cracking of fruits and AaBAM3 gene is the main β-amylase in the starch degradation process of fruits. Studies have reported that in Arabidopsis thaliana leaves and potato tubers, BAM3 gene is induced by cold, which is contrary to the results in the Annona fruit. However, AaBAM3 has not been reported in Atemoya and the transcriptional regulation mechanism of BAM3 gene in response to low temperature in plants is still unclear at present. We will (1)clone the promoter sequence of AaBAM3 gene and analyze cis-acting elements; (2) establish the yeast one-hybrid cDNA library of Atemoya fruit pulp under cold treatment and screen transcription factors by using yeast one-hybridnd; (3) analyze the expression of transcription factors at the level of transcription and protein; (4) verify transcription factors by EMSA and ChIP-qPCR; (5) examine its regulation by dual-luciferase system. In order to explore the transcriptional regulation mechanism of AaBAM3 in fruit starch degradation. In this project, these results will help to understand the transcriptional regulation mechanism of starch degradation in fruits of higher plants, and will be useful for extending the shelf life of Atemoya fruit.

淀粉是番荔枝果实中主要的碳水化合物。前期研究发现淀粉降解直接关系果实的软化和开裂，AaBAM3是果实淀粉降解过程中主要的β-淀粉酶基因，同时还发现低温处理抑制果实中AaBAM3表达，这与在拟南芥叶片、马铃薯块茎中BAM3受低温诱导的结果相反。目前尚未有关于番荔枝AaBAM3的研究报道，BAM3在植物中响应低温的转录调控机制尚未明确。本项目拟克隆AaBAM3基因的启动子序列，分析顺式调控元件；建立冷处理下果肉酵母单杂交cDNA文库，利用酵母单杂交、回杂，筛选与之结合的候选转录因子；从转录和蛋白水平对候选转录因子进行表达分析；利用EMSA和ChIP-qPCR技术进行体外和体内验证；通过双荧光素酶体系研究其对AaBAM3的调控方式，从而探明AaBAM3在果实淀粉降解中响应低温的转录调控机制。该研究能够为高等植物果实淀粉降解的转录调控机制提供理论支持，还可为延长番荔枝果实货架期提供理论依据。

番荔枝是一种优稀特色水果，其存在采后货架期短，夏季果容易裂果的问题。淀粉是番荔枝果实中主要的碳水化合物。前期研究发现淀粉降解的速度直接关系果实的软化和开裂程度，低温处理下，淀粉降解减缓，AaBAM3表达水平较常温抑制表达。本研究旨在探明AaBAM3在果实淀粉降解中响应低温的转录调控机制，通过研究发现番荔枝AaBAMs基因家族中，AaBAM3无明显组织特异性表达，但是果实后熟过程特异诱导表达，AaBAM3除了在低温下抑制表达，其受乙烯及低湿条件诱导表达，与淀粉含量和β-淀粉酶活性变化进程一致，进一步说明AaBAM3是果实淀粉降解过程中主要的β-淀粉酶基因。亚细胞定位发现其定位在叶绿体，与颗粒淀粉合成酶AtGBSS蛋白位置可以重合，表明其定位在淀粉颗粒上。克隆了AaBAM3启动子并分析其顺式调控元件，通过双荧光素酶实验，解析了其核心调控区域，并通过酵母单杂交实验筛选到与AaBAM3启动子结合的两个转录因子AaHAT22和AaDIV。通过酵母回杂交、DAP-seq和EMSA实验分别验证了其与AaBAM3启动子存在结合，双荧光素酶实验分析发现AaHAT22、AaDIV对AaBAM3启动子起抑制作用。推测番荔枝果实低温处理下，AaHAT22、AaDIV通过抑制AaBAM3的表达，延缓了淀粉降解，从而推迟成熟进程，该研究能够为果实淀粉降解的转录调控机制提供理论支持，还可为延长番荔枝果实货架期提供理论依据。