茶树花粉管抗寒相关类钙调蛋白CsCML21基因及启动子功能分析

Considering the phenomena that tea pollen tube can elongate at low temperature but cannot continue to finish the fertilization, we screened and cloned Calmodulin Like Protein (CML) gene CsCML21 which was primarily verified to be related to the cold tolerance of pollen tube. In order to understand the involvement of CsCML21 in cold-tolerant-ability of tea pollen tube, this new project is designed to research the following works: cloning cDNA full length of CsCML21 using RACE, and the sequence of its promter (pCsCML21) using TAIL-PCR; analyzing the sequences both of CsCML21 and pCsCML21 in bioinformatics, and identifying the functional elements of Ca2+ and ABA in pCsCML21; verifying the functional elements and the regulation of Ca2+ and ABA on the promoter using site-directed mutation method with the constructed expression vector of pCsCML21:GUS transformed to Arabidopsis; constructing the expression vector of pCsCML21:CsCML21 and transforming it to Arabidopsis for the functional analysis of CsCML21. The results from the research work mentioned above can also provide the scientific theory basis for the study on the mechanism of tea self-incompatibility and the breeding for cold tolerant tea cultivars.

针对茶树花粉管在低温下能够生长而受精困难的科学问题，我们以花粉管为切入点，筛选并克隆了CsCML21基因，初步研究发现该基因与茶树花粉管抗寒性有关。为了更全面的了解该基因参与茶树花粉管抗寒性的机理，本项目拟开展以下研究工作：分别利用RACE技术和TAIL-PCR法克隆其cDNA全长和其启动子pCsCML21；对CsCML21和pCsCML21序列进行生物信息学分析；利用定点突变的方法寻找启动子中Ca2+和ABA的作用元件，构建pCsCML21:GUS表达载体，转入拟南芥，分析Ca2+和ABA对CsCML21启动子的诱导表达作用；构建pCsCML21：CsCML21表达载体并将其转入拟南芥进行表达，验证CsCML21功能。该项目的完成同样为茶树自交不亲和的研究和抗寒品种选育提供科学依据。

类钙调蛋白（Calmodulin like protein，CML）是一类植物细胞所特有的Ca2+响应蛋白家族，在花粉萌发和花粉管生长过程中起着重要的调控作用。本研究用cDNA-AFLP技术获得CsCML21序列片段，并利于RACE技术获得基因全长序列。序列比对显示CsCML21与其他物种同源基因序列具有很高的相似性，其推导氨基酸序列与与葡萄VvCML21亲缘关系最近。亚细胞定位结果表明CsCML21主要表达于细胞质中。QRT-PCR分析发现CsCML21在花粉中表达量最高，且可以响应低温信号。根据CsCML21基因序列，利用Tail-PCR得到长为1739bp的启动子序列。pCsCML21序列含有启动子典型的TATA框及CAAT-box。在启动子的上游发现了两个Ca2+/ABA响应的作用元件ABRE。利用拟南芥验证启动子活性，结果表明ABRE元件的缺失削弱了pCsCML21启动子的活性。为了深入研究CML基因家族在茶树逆境响应中的作用机制，根据转录组序列获得5条CsCML21旁系同源基因序列，qRT-PCR结果表明不同茶树CML基因在组织中表达量的差异，预示着CML基因家族在茶树体内可能具有不同的功能分化，且参与非生物胁迫的响应。此外，利用RACE和RT-PCR技术克隆获得CsCML45。功能分析发现，过表达CsCML45增强了转基因拟南芥的耐热性。CsCML21基因、启动子及其旁系同源基因的功能分析为研究茶树花粉生长及逆境响应提供了重要的理论依据。