苹果sMdCAX11基因的功能分析与鉴定  

Functional analysis and characterization of the sMdCAX11 gene in apple

作　　者：刘佳 殷伟杰 李宇坤 王彩霞[1] 任小林[2] LIU Jia;YINWeijie;LI Yukun;WANG Caixia;REN Xiaolin(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;College of Horticulture,Northwest Agriculture and Forestry University,Yangling 712100,Shaanxi,China;Mengniu Hi-tech Dairy Product Beijing Co.,Ltd.,Beijing 101100,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]西北农林科技大学园艺学院,陕西杨凌712100 [3]蒙牛高科乳制品(北京)有限责任公司,北京101100

出　　处：《果树学报》2024年第7期1275-1284,共10页Journal of Fruit Science

基　　金：国家自然科学基金项目(32300327)。

摘　　要：【目的】深入分析苹果sMdCAX11(去掉NRR区域的MdCAX11)基因功能。【方法】分别利用蜜脆苹果果实和拟南芥材料,采用过表达sMdCAX11的试验方法,观察果实、叶片等各组织的表型,并测定不同组织的矿质元素含量,同时对苹果MdCAX11基因启动子区域进行预测分析以及启动子转录活性分析。【结果】瞬时过表达sMdCAX11的苹果果肉颜色变褐,并出现皱缩;同时过表达sMdCAX11的苹果果肉和拟南芥叶片的总Ca含量明显下降,且元素比值(K+Mg)/Ca明显升高。【结论】sMdCAX11基因过表达可导致植株组织的元素分配不均,不同元素间的比例失衡。【Objective】Ca^(2+)/H^(+)antiporter(CAX)is a type of low-affinity and high-capacity transporter that mainly relies on the transmembrane proton gradient to complete the transport of Ca^(2+).This protein may be related to the occurrence of plant calcium deficiency.It is known that Ca^(2+)/H^(+)reverse transporter proteins(CAXs)in model plants like Arabidopsis thaliana and tomato play important roles in regulating intracellular Ca^(2+)distribution and allocation,and maintaining intracellular calcium homeostasis,and overexpression of the CAXs genes in different plants could cause calcium deficiency symptoms in the plants.In the preliminary stage of this study,Honeycrisp apple bitter pit disorder fruits with different degrees of incidence were used as test materials,and the differences in mineral element contents and expression patterns of calcium transport-related genes in disordered fruits were analyzed.The key regulatory genes MdCAX5 and sMdCAX11(MdCAX11 with the N-terminal autoinhibitory region removed)involved in the development of bitter pit disorder were then identified.However,the function of Md-CAX11 protein in apple was still unclear.Meanwhile,it was still unclear whether Ca^(2+)/H^(+)reverse transporter proteins were involved in the development of bitter pit disorder in fruit.【Methods】The gene functions of sMdCAX11 were analyzed using experimental methods like genetic transformation.In this study,we first utilized the transient transformation of Honeycrisp apple fruits to verify the calcium transport function of the sMdCAX11 protein.Fruits transiently transformed with the sMdCAX11 gene were sectioned to observe the changes in the flesh tissue near the injection hole,and the mineral element content of the flesh tissue was also determined.Next,we stably transformed sMdCAX11 into the Arabidopsis Col-0 and successfully obtained positive T4 generation transgenic plants.The PCR tests at DNA level and RNA level verified that all obtained were positive plants,and the leaves were analyzed for mineral element

关 键 词：苹果 Ca^(2+)/H^(+)反向转运体(CAX) 基因功能 矿质元素 钙含量 

分 类 号：S661.1[农业科学—果树学]