紫花苜蓿MsCCD4基因克隆及耐盐功能鉴定

为验证紫花苜蓿(Medicago sativa L.)胡萝卜素裂解双加氧酶(Carotenoid cleavage dioxygenase，CCD)基因的耐盐功能，本试验克隆了紫花苜蓿MsCCD4的CDS全长序列，并以其叶片为试验材料，通过发根农杆菌介导的毛状根诱导法获得过表达MsCCD4的转基因毛状根，验证过表达MsCCD4对紫花苜蓿耐盐性的影响。结果表明：MsCCD4为稳定的酸性亲水蛋白质，二级结构以无规则卷曲为主，C-端含有RPE65保守结构域；MsCCD4启动子区含有光响应、激素应答和非生物胁迫响应结合元件；盐胁迫可诱导MsCCD4表达量升高；盐胁迫生理实验证明，过表达MsCCD4可以增强毛状根的耐盐性。本研究为紫花苜蓿耐盐遗传改良提供基因资源及耐盐基因快速鉴定方法。

Cloning and Identification of Salt Tolerance Function of alfalfa MsCCD4

To verify the salt tolerance function of Carotenoid cleavage dioxygenase (CCD) gene of alfalfa,we cloned the full-length CDS sequence of alfalfa MsCCD4. By using bioinformatics method analysis,we discovered that MsCCD4 protein is an acidic hydrophilic protein with an irregular curl and conservative RPE65 domain at the C-terminus. Cis-regulatory elements of the MsCCD4 promoter include elements involved in light-responsiveness,hormone response,and abiotic stress. The expression of MsCCD4 significantly responds to salt stress. Overexpression of MsCCD4 transgenic hairy roots was obtained by Agrobacterium rhizogenes- mediated hairy root induction from leaf explant of 'Zhongmu No.1' alfalfa. The results showed that overexpression of MsCCD4 enhanced the salt tolerance of the transgenic hairy roots. The study provides an effective method for salt-tolerant gene functional determination of alfalfa and provides gene resources for salt tolerance improvement of alfalfa.