大白菜KIN基因的表达及其pre-mRNA加工机制研究

Low temperature is one of the most important abiotic factors limiting productivity and quality of Brassica campestris L.ssp.pekinensis. Cold-regulated (COR) genes can be induced to express by cold stress, and play a key role in conferring freezing/cold tolerance of plants. Post-transcriptional regulation is one of the important stages in regulating the expression of COR genes. Due to whole genome sequencing of Brassica rapa (Chiifu-401), it is convenient and important for analyzing the expression and pre-mRNA processing mechanism of the cold-regulated KIN genes from the antifreeze / not antifreeze materials of Brassica campestris L.ssp.pekinensis. By RACE combining with PAGE, we can analyzed the length polymorphism of 3'UTR of the KIN transcripts for checking the sites and frequency of polyadenylation of the KIN genes from Brassica campestris L.ssp.pekinensis. Further, the different spliced transcripts of the KIN genes, produced from the same pre-mRNA by alternative splicing, are obtained and sequenced for analyzing the spliced sites of the KIN genes by PCR with specific primer sets. We analyzed comparatively the differences of polyadenylation of the KIN genes in pre-mRNA processing between different varieties of Brassica campestris L.ssp.pekinensis, and characterized the pre-mRNA processing obviously related to cold tolerance. Based on the former study, the characteristics of pre-mRNA processing of the KIN genes are determined respectively when seedlings are subbject to hormones and stress treatment. Finally, we will demonstrate the main transcripts, processed by alternative splicing and alternative polyadenylation, are involved in conferring cold tolerance of plants. It is very significant to understand the relationship between pre-mRNA processing mechanisms and abiotic stresses, and the result of this study will provide a theoretical basis for the next step to determine downstream biological function of the KIN transcripts processed by the pre-mRNA processing

持续的冻害会引起大白菜产量和品质的下降，抗冻基因的诱导表达对保护其渡过低温环境起着关键作用，转录后加工是调控抗冻基因表达的重要阶段。本项目以白菜全基因组测序为基础，以抗/不抗冻的大白菜为材料，研究抗冻基因KIN的表达及其pre-mRNA加工特点。通过RACE结合PAGE技术，分析KIN基因的3'UTR的多态性，鉴定多聚腺苷酸化位点及频率。通过特异引物进行PCR，鉴定KIN的不同剪接位点。比较分析抗冻/不抗冻大白菜种质间KIN基因的pre-mRNA加工的差异，找出与大白菜抗冻性密切相关的主要pre-mRNA加工特征。在此基础上，对大白菜在不同激素和胁迫处理下KIN基因的pre-mRNA加工特点深入分析，确定KIN基因发挥抗冻功能的主要转录后加工产物。该研究结果对了解大白菜抗逆基因转录后加工机制与胁迫之间的关系具有重要意义，为下步开展KIN的pre-mRNA加工产物的下游生物学功能研究提供基础

一、研究背景.大白菜在我国的蔬菜生产和消费中占有极其重要的地位，而每年由低温造成的减产时有发生。冷调控（COR）基因的诱导表达能增强大白菜对低温的抵抗能力。KIN是COR家族基因中的重要成员，研究发现拟南芥的KIN基因参与了对低温、盐胁迫及干旱胁迫的响应。随着白菜基因组测序完成，可帮助我们研究大白抗冻基因KIN的选择性剪接和选择性多聚腺苷酸化、起源及其与胁迫之间的关系。.二、研究内容.1、大白菜KIN基因家族成员的克隆、测序、鉴定以及pre-mRNA加工的预测.2、大白菜KIN基因pre-mRNA的选择性剪接和选择性多聚腺苷酸化识别和鉴定.3、大白菜KIN基因的pre-mRNA的特殊加工机制与胁迫之间的关系及其起源分析 .三、重要结果.1、白菜和甘蓝中均有2个KIN基因，分为KIN1类和KIN2类。在转录水平上，BrKIN1广泛表达，而BrKIN2特异表达在雄蕊中。ABA和低温处理叶片后，BrKIN1会显著上调，而BrKIN2不表达。.2、BrKIN1的选择性剪接广泛存在，而BrKIN2的选择性剪接仅存在于雄蕊中。BrKIN1和BrKIN2存在选择性多聚腺苷酸化，其中BrKIN1的剪接异构体中均存在着多个选择性多聚腺苷酸化位点，且选择性多聚腺苷酸化广泛存在于多种发育时期与胁迫条件下。.3、KIN基因这种pre-mRNA加工机制是极其保守的，且在十字花科植物的共同祖先分化之前可能就已经存在。通过对BrKIN1基因的剪切异构体的转录水平分析，发现ABA和低温也可以诱导这种选择性剪接异构体的表达。.四、关键数据.白菜中的BrKIN1和BrKIN2分别定位在A02和A10染色体上。两者在氨基酸序列上高度保守，是富含丙氨酸的一类碱性蛋白。BrKIN1和BrKIN2在启动子区域均存在一个“ABRE-LTR-ABRE”的基序，其中BrKIN2的ABRE核心序列5’-ACGTG-3’突变成了5’-ACTTG-3’。BrKIN1和BrKIN2分别存在4种和3种剪接异构体，BrKIN1至少存在9个poly（A）位点，而BrKIN2至少存在2个。.五、科学意义.本项目确定大白菜KIN基因pre-mRNA加工基本特点。该项目的完成预期能够获得大量的包含可变3’UTR的质粒与多种选择性剪接异构体的质粒，为后续分析不同3’UTR和不同剪接异构体对蛋白质翻译效率的影响夯实基础。