内含子低温诱导型增强子调控马铃薯块茎VInv基因表达的分子机理

Potato vacuolar invertase gene (VInv) is highly expressed in cold-stored tubers and its expressed product vacuolar invertase could hydrolyze sucrose to reducing sugars. Moreover, reducing sugars could react with asparagine and produce carcinogen acrylamide, which is a hazard to human health, in processed potato products during high-temperature processing. Previous studies mainly focused on silencing of the VInv gene and suppression of the enzyme activity, however the reason for highly expression of the VInv gene response to cold stress in tubers is still unclear. Our previous studies demonstrated that the second intron of the VInv gene had strongly enhancer function and was cold inducible in cold-stored tubers, so we proposed that the intronic enhancer might regulate the VInv gene expression under cold stress. In this project, tRNA-CRISPR/Cas9 genome editing system would be used to verify that if the enhancer could affect the VInv gene expression, and Chromosome Conformation Capture Combined with sequencing (4C) would be conducted to detect the promoter element that interacts with the intronic enhancer of the VInv gene. Then yeast-one-hybrid and yeast-two-hybrid analysis would be used to identify transcription factors binding to the enhancer or promoter and proteins interacted with transcription factors, respectively. These results would expand our understanding for regulatory mechanism of the VInv gene expression under cold stress, which will finally benefit for potato resistance or tolerance breeding on cold-induced sweetening.

马铃薯低温贮藏时，块茎中的液泡酸性转化酶基因VInv高度上调表达，产生的液泡酸性转化酶能将蔗糖水解成还原糖，在高温时与天冬酰胺起反应产生致癌物质丙烯酰胺，严重威胁到人类健康。以往的研究大多集中于VInv基因表达的下游机制，包括对转录产物的干扰和翻译产物活性的抑制等，针对基因表达上游调控通路的研究有待于进一步深入。申请人前期研究发现，VInv基因的第二个内含子具有增强子的功能并且对低温强烈响应，推测可调控VInv基因表达，但机理尚不明确。本课题通过tRNA-CRISPR/Cas9基因组编辑技术验证增强子对VInv基因表达的影响，并利用染色体空间构象捕获测序技术定位与增强子互作的VInv基因启动子元件，进而利用酵母单、双杂交技术筛选与增强子或启动子结合的转录因子以及与转录因子互作的蛋白，以明确VInv基因受低温诱导表达的调控机制。研究结果可为抗/耐低温诱导糖化育种提供理论依据和基因资源。

马铃薯低温贮藏时，块茎中的液泡酸性转化酶基因VInv高度上调表达，产生的液泡酸性转化酶能将蔗糖水解成还原糖，在高温时与天冬酰胺起反应产生致癌物质丙烯酰胺，严重威胁到人类健康。以往的研究大多集中于VInv基因表达的下游机制，包括对转录产物的干扰和翻译产物活性的抑制等，针对基因表达上游调控通路的研究有待于进一步深入。申请人前期研究发现，VInv基因的第二个内含子具有增强子的功能并且对低温强烈响应，推测可调控VInv基因表达，但机理尚不明确。本项目以600bp增强子DNA元件为基础序列，通过构建14个增强子功能验证载体（RH11～RH24），结合拟南芥异位功能分析方法，将增强子核心DNA元件定位到200bp的区间范围内。接着以200bp增强子核心DNA元件为目标，构建一个由2×35S启动子驱动Cas9蛋白基因和分别由AtU3或AtU6启动子驱动4个不同gRNA的pHN-VInv融合编辑载体，通过农杆菌介导法转化马铃薯，获得8个大片段剪切缺失系，验证了该增强子对VInv基因本身具有调控作用。同时，生物信息学分析发现，该200bp增强子核心DNA元件含有bHLH、MADS、HMGA等14类Motif，通过无效序列逐个替换单类Motif的策略，结合拟南芥异位功能分析方法，获得2个与增强子相关的潜在的Motif（G2-Like和TCP）。进而利用染色体空间构象捕获技术定位到与200bp增强子互作的“潜在启动子区域”。以串联3个重复的增强子或启动子作为诱饵序列，通过酵母杂交及相关验证试验鉴定出与增强子和启动子分别存在显著互作的2个阳性蛋白，初步明确了VInv基因受低温诱导表达的调控机制。研究结果可为抗/耐低温诱导糖化育种提供理论依据和基因资源。