甘蓝BoSWEET11c和BoSWEET12b响应低温胁迫的功能分析

In view of the important role of temperature during the growth and development in cabbage, focusing on the scientific problem of how BoSWEETs respond to low-temperature stress. A total of 30 BoSWEET genes were identified in previous study, in which BoSWEET11c and BoSWEET12b expressed in leaves in response to low-temperature stress, with its promoter sequences having three and one low-temperature response elements LTRE, respectively. It was speculated that BoSWEET11c and BoSWEET12b are critical genes in response to low-temperature stress of cabbage. This study was to analyze the correlation between different cold resistance and the differential sites of SNP/Indel in BoSWEET11c and BoSWEET12b promoter sequences of cabbage, and to analyze the correlation between the soluble sugar content and the expression level of BoSWEET11c and BoSWEET12b in different organs. The coding sequence and promoter sequence of BoSWEET11c and BoSWEET12b were isolated and the dual luciferase reporter system was used to verify whether BoSWEET11c and BoSWEET12b induced by low temperature. The over-expression and inhibition expression vectors of BoSWEET11c and BoSWEET12b were constructed, and the subcellular localization and genetic transformation were performed for functional verification. The results will help to analyze the molecular function in response to low-temperature stress, and provide an important theoretical basis for the genetic improvement of chilling tolerance traits in cabbage.

针对温度在甘蓝生长发育过程中的重要作用，围绕BoSWEETs如何响应低温胁迫这一科学问题，我们前期鉴定了30个BoSWEETs基因，其中BoSWEET11c、12b在叶片中的表达响应低温胁迫，并且其启动子中分别含有3个和1个低温响应元件LTRE，据此推测BoSWEET11c、12b是甘蓝响应低温胁迫重要基因。本研究拟分析甘蓝不同抗寒性与BoSWEET11c、12b启动子序列SNP/Indel差异位点的相关性，分析甘蓝不同器官可溶性糖含量与BoSWEET11c、12b表达量的相关性；分离克隆BoSWEET11c、12b的CDS和启动子序列，利用双荧光素酶报告系统验证BoSWEET11c、12b是否受低温诱导；构建BoSWEET11c、12b的过表达与抑制表达载体，进行亚细胞定位以及遗传转化进行功能验证。研究结果将有助于解析甘蓝响应低温胁迫的分子功能，为甘蓝耐寒性状遗传改良提供重要理论基础。

低温是影响甘蓝生长发育、产量和品质的主要逆境因子之一。SWEET蛋白是高等植物中一类跨膜转运糖的易化扩散蛋白家族，参与光合同化物从“源”到“库”的质外体装载运输过程。低温胁迫下拟南芥双突变体atsweet11/atsweet12表现出韧皮部装载缺陷表型，叶片中有大量蔗糖积累，使得其耐寒性明显高于野生型。然而甘蓝中BoSWEETs基因如何响应低温胁迫尚无报道。.本项目前期在甘蓝基因组数据库中鉴定出30个BoSWEET基因成员，通过低温胁迫筛选出2个关键基因BoSWEET11c和BoSWEET12b。在此基础上，通过对20份耐寒甘蓝和20份不耐寒甘蓝中BoSWEET11c和BoSWEET12b基因启动子和编码序列进行变异位点检测，共鉴定出71个变异位点，但未找到与耐寒性相关的变异位点。测定了低温胁迫后不同耐寒性甘蓝叶片中BoSWEET11c和BoSWEET12b基因的表达量以及叶片中不同糖类含量，发现耐寒甘蓝‘CT-923’叶片中BoSWEET11c和BoSWEET12b基因在低温胁迫24h时处于低表达水平，同时其叶片中蔗糖含量升高，即BoSWEET11c和BoSWEET12b基因的低表达使得转运到韧皮部的蔗糖减少，导致叶片中蔗糖含量增加。亚细胞定位分析表明BoSWEET11c和BoSWEET12b蛋白均为细胞膜定位。构建了BoSWEET11c、BoSWEET12b和BoSWEET11c-12b基因过表达载体并遗传转化拟南芥，发现BoSWEET11c、BoSWEET12b和BoSWEET11c-12b转基因植株的冻死率远远高于野生型。推测是BoSWEET11c和BoSWEET12b基因的高水平表达，使得转基因植株叶片中蔗糖含量大量减少，叶片中没有足够的蔗糖来抵御低温造成的伤害。上述研究结果为进一步解析甘蓝响应低温胁迫的分子功能以及耐寒性状遗传改良提供了重要理论依据。