ALAD生化特性、编码基因克隆及其与香蕉抗寒性关系的研究

Banana is one of the most important tropical fruits in South China. It is often injuried by cold stress, which has become one of the restriction factors for banana's sustainable development. In our former research, we have ascertained that ALA can promote the chilling tolerance of banana seedlings. As a key enzyme of ALA metabolism, reserchers home and abroad focus on ALAD in animals and medical,while less attention has been paid to study in plant. The function of ALAD on the enviromental adaptation of plants, especially in bananas remain unclear. In our study, ALAD gene of banana will be cloned by PCR and RACE, then be transformed into Arabidopsis to study the physiological response to low temperature stress. Meanwhile, with chromatographic technique, such as superdex,DEAE Sepharo se CL-6B and Phenyl Sepharose CL-4B,banana ALAD will be separated and purificated to study its characteristics, then after being pretreated with ALAD,the physiological and biochemical changes of banana under chilling stress will be detected to explore the regulation mechanism of ALAD on chilling resistance of banana. The results will be helpful for elucidation of ALAD regulation theroy and improvement of chilling tolerance in banana.

香蕉是我国热带地区重要热带水果，低温寒害已成为香蕉生产可持续发展的主要限制因子之一。本课题组前期研究表明，ALA可以有效调节香蕉的抗寒能力，ALAD作为ALA代谢的关键酶在动物、人体医学上的研究较为透彻，但对植物ALAD的研究却相对缺乏。ALAD在植物低温逆境适应中的作用与功能至今尚不清楚。本项目利用PCR 和RACE技术克隆香蕉ALAD基因全长，并将该基因转化到拟南芥体内，以研究转化植株对低温逆境的生理响应。同时，利用双水相萃取技术、superdex、DEAE Sepharo se CL-6B、Phenyl Sepharose CL-4B等层析技术分离纯化香蕉ALAD，分析其相关酶学特性，并研究过量表达ALAD基因香蕉及ALAD抑制剂处理后香蕉的抗寒生理及相关基因的动态变化，以探索ALAD对香蕉抗寒性的内在调控机理。本研究可为拓展研究香蕉ALAD的生理功能、基因表达与调控提供理论基础。

香蕉是我国热带地区重要热带水果，低温寒害已成为香蕉生产可持续发展的主要限制因子之一。本项目利用PCR 和RACE技术克隆获得两个品种香蕉ALAD基因全长，分析了该基因的生物学信息，并将该基因转化到拟南芥和大肠杆菌，获得转香蕉ALAD基因拟南芥植株和原核表达菌株，优化了香蕉ALAD原核表达技术体系：在28℃下，0.5mM 诱导剂IPTG诱导1h后再常规培养6h。利用镍琼脂糖亲和层析、SDS-PAGE技术分离、纯化香蕉ALAD蛋白，分析了该ALAD蛋白的基本酶学特性，发现该酶是一个金属依赖酶，对Mg2+依赖程度最大，其在PH8.5、37℃条件下活性最高，乙酰丙酸与巯醇皆会抑制其活性。此外，本项目还从转化拟南芥植株的抗寒性表型、香蕉ALAD基因对低温逆境的表达响应、外源喷施香蕉ALAD蛋白三方面研究了香蕉ALAD与香蕉抗寒性之间的关系，发现在低温逆境胁迫下，香蕉叶片ALAD基因会表达量会成倍增加，转基因拟南芥过量表达香蕉ALAD时，其抗寒力与恢复能力显著增强，外源喷施预处理香蕉ALAD蛋白会提升香蕉内ALAD活性，同时还显著提升POD、SOD活性，电导率、膜脂透性（MDA含量）均显著降低，可溶性蛋白含量、脯氨酸含量显著增加，增强了细胞对水分的束缚能力和叶绿素的维持能力，细胞相对含水量与叶绿素含量显著提高，从而增强了香蕉的低温适应能力。本研究可为拓展研究香蕉ALAD的生理功能、基因表达与调控提供理论基础。