花生种子特异启动子AHSSP1的克隆及功能分析

为丰富花生种子特异启动子资源,本研究利用PCR技术在花生基因组中克隆了种子贮藏蛋白基因PSC32的启动子AHSSP1,利用半定量RT-PCR检测了PSC32基因表达模式,借助NewPLACE在线分析了AHSSP1序列中存在的顺式作用元件,并构建了AHSSP1驱动GUS报告基因的表达载体,经农杆菌转化获得转基因拟南芥,经GUS组织化学染色鉴定了该启动子的功能。结果表明,PSC32基因957 bp长的启动子AHSSP1序列具备种子特异表达启动子特有的3个RY REPEAT元件。半定量RT-PCR分析发现,PSC32基因在花生成熟种子中表达,而在饱果成熟期根、茎、叶片、花、入土前的果针、成熟种子的果壳中均不表达。GUS组织化学染色发现,转基因拟南芥成熟种子以及萌发种子的子叶、下胚轴和胚根均能够被染上蓝色;长出真叶后,子叶和下胚轴仍能被染色,而根和真叶不能被染上蓝色;成年期转基因拟南芥的叶片也不能被染上蓝色。而野生型拟南芥整个生长时期均不能被染上蓝色。以上现象说明AHSSP1是一个种子特异启动子。本研究丰富了花生种子特异启动子的资源,对花生籽仁品质改良或以花生籽仁作为“生物反应器”的研究具有重要的应用价值。

Isolation and Functional Analysis of Seed Specific Promoter AHSSP1 From Peanut(Arachis hypogaea L.)

To enrich the resources of seed specific promoters, we PCR-amplified the promoter AHSSP1 of seed storage protein PSC32 from peanut genome. The expression patterns of PSC32 gene was detected by semi-quantitative PCR, cis-acting elements in AHSSP1 were analyzed using online NewPLACE. GUS gene driven by AHSSP1was transformed into Arabidopsis. The function of AHSSP1 was characterized through GUS histochemical staining. Results showed that AHSSP1 ahd 957 bp in length, with 3 RY REPEAT, which were commonly dispersed in seed specific promoters. PSC32 gene was found to be expressed specifically in seed, but not expressed in root, stem, leaf, flower, peg, and pod shell of mature seed in pod maturing stage based on semi-quantitative PCR analysis. Histochemical staining of Gus activity showed that mature seeds of transgenic Arabidopsis, and the cotyledons, hypocotyl and young root of germinating seeds were all stained blue. Cotyledon and hypocotyl still exhibited GUS staining activity after growing true leaves, while the roots and true leaves did not display GUS staining activity. The leaves of adult transgenic Arabidopsis could not be stained blue, while non-transformed Arabidopsis could not be stained blue throughout the growth period. It indicated that AHSSP1 was a seed specific promoter. This study enriched the resources of peanut seed specific promoters, and would play important application in improving peanut kernel quality or using peanut seed “bioreactor” research.