Copy detection and expression profile analysis of FAD2 genes in Caragana intermedia | 中间锦鸡儿FAD2基因拷贝数检测及组织表达谱分析

Chino. 探索中间锦鸡儿基因组中FAD2基因的拷贝数，分析不同拷贝的表达模式，以期揭示不同拷贝在植物体内的功能分工。根据中间锦鸡儿（Caragana intermedia）FAD2基因的保守序列设计1对引物SF和SR，利用该引物对PCR扩增制备114 bp的Southern检测探针，探针进行地高辛标记后，采用罗氏Southern杂交试剂盒进行Southern检测。根据中间锦鸡儿3个FAD2基因各自的特异差异序列，分别设计定量PCR引物序列，以actin基因为内参，对中间锦鸡儿的根、茎、叶及不同发育时期（发芽15，25，35 d）种子的cDNA进行定量PCR分析。中间锦鸡儿FAD2基因至少有4个拷贝，且不同拷贝的表达模式不同。FAD2-2A基因在根和发育中期、后期的种子中低水平表达，在幼嫩的茎、叶以及发育早期的种子中高水平表达；FAD2-1A基因在根中的表达量最低，在种子发育早期、中期大量表达，在幼嫩叶子中表达水平也较高；FAD2-1B基因仅在种子发育中期大量表达，在其他组织及种子其他发育阶段的表达量均保持在本底水平。在不同组织以及种子的不同发育时期，FAD2-1A相对表达量的变化幅度最大，达到了近100倍，FAD2-1B次之，FAD2-2A最小。结合序列比对分析推测：FAD2-2A基因可能主要负责合成膜脂中的亚油酸，FAD2-1A主要负责种子及叶子贮脂中亚油酸的合成，FAD2-1B负责种子贮脂中油酸的去饱和作用。

Inglés. The study was to detect the FAD2 gene copies, analyze the FAD2 gene expression profile and presume every copy’s function in Caragana intermedia. The Southern-blotting probe was prepared by PCR with the primers designed according to the consensus sequence of FAD2 genes in C. intermedia. The probe was labeled by digoxin and Southern-blotting was done with Roche Southern-blotting kit. The real-time PCR primers were designed according to the distinct sequences of every FAD2 copy. The quantity PCR was done for root, stem, leaves and the young seed of C. intermedia with the actin gene as interior control. Southern-blotting results indicated there were four copies of FAD2 gene in C. intermedia. genome at least, and these were coincident with the four FAD2 genes cloned. The real-time PCR was performed to detect the relative expression levels of FAD2-2A, FAD2-1A and FAD2-1B mRNA. FAD2-2A transcript showed a low level in the root, middle-stage seeds and pre-mature seeds, and FAD2-2A was expressed more in the first-stage seeds and tender leaves than in the other tissues. FAD2-1A transcripts showed the lowest level in the root and the highest level in the first-stage and middle-stage seeds, and it was expressed abundantly in the tender leaves, too. FAD2-1B was expressed abundantly in the middle-stage seeds and its transcript was very low in the other tissues.In all tissues the FAD2-1A transcript level changed most, and that of FAD2-2A changed least. According to all of these, we deduced that FAD2-2A takes responsibility for desaturating oleic acid in the membrane lipid mainly; FAD2-1A is in charge of desaturating oleic acid in the store lipid of seeds and leaves; and FAD2-1B is in charge of desaturating oleic acid in the store lipid of seeds, too.