摘要
在植物的许多发育阶段,MADS-box家族基因都扮演了重要的角色。它控制了植物的花器官发育,在植物侧根,果实,种皮的发育过程中也起了很重要的作用。拟南芥中报道的MADS-box基因有107个,而在大豆中还知之甚少。GmMADS28及GmAGL15是MADS-box家族基因两个重要成员,GmMADS28在花中优势表达,序列分析和系统发生分析表明是SEP亚家族成员,GmAGL15在幼胚中表达量较高。本研究对这两个基因进行了初步的研究。
为了研究GmAGL15基因功能,首先本实验利用洋葱表皮对GmAGL15进行了亚细胞定位,发现其定位于细胞核和细胞膜。对其进行序列比对与系统发生分析,发现GmAGL15与拟南芥的AGL15相似性较高,达69%。该基因的基因组结构的分析表明它们都含有7个内含子,蛋白的N,C末端都含有一段保守序列,推测GmAGL15基因与拟南芥AGL15同源。
为探明大豆GmAGL15基因的表达调控规律,应用电子克隆技术从大豆基因组中克隆了GmAGL15翻译启始位点上游1000bp的DNA片段,用PLACE在线启动子预测工具分析表明:该序列含有启动子的特定结构,如TATA-BOX、CAAT-BOX。另外还含有一些顺式作用元件,如调控GmAGL15的组织特异的和特定发育阶段的表达,对胁迫的应答,光调节,以及反馈调节,推测大豆GmAGL15基因表达有相应组织特异性,可能受蔗糖、生长素和乙烯等的调控。用PLACE在线启动子预测工具对大豆与拟南芥等其他4种植物的同源基因启动子的顺式元件进行统计比较,发现不同植物的启动子既有保守性,又有多样性,转录因子结合位点的分布相似,但也有区别,暗示了GmAGL15基因表达调控的精确性或多样性。
本研究还初步进行了GmAGL15基因在烟草中的异位表达,并对转化植株的基因表达进行了荧光定量检测,表明该基因得到了表达,但是植株与对照相比无明显表型变化。
为了研究GmMADS28基因功能,本实验利用农杆菌介导法初步进行了GmMADS28基因在大豆中的增强表达,获得了62株抗性植株,发现少数有提早开花现象。同时应用Gateway技术体系替代传统载体的构建方法,并结合近年来广泛应用于植物基因功能研究的RNA干涉技术,构建了大豆基因GmMADS28的RNA干涉载体,并转化了根癌农杆菌,为下一步的转基因研究奠定了基础。
MADS-box genes are known to be involved in many important processes during plant development.They are key components regulating flowering transition and play an important role during the development of lateral root,fruit and seed-coat.Genome-wide identification of MADS-box genes has revealed I07 genes in Arabidopsis,but little is known in the soybean.GmMADS28 and GmAGL15 are two important members of MADS-box genes family.GmMADS28 has high expression in flowers.Analysis of sequence alignment and phylogenetic tree indicated that it belongs to SEP subfamily. GmAGL15 expresses only in embryos.This paper made primary research on the two MADS-box genes.
In order to investigate the function of GmAGL15,subcellular localization by transient expression of GmAGL15-GFP fusion protein on onion epiderm showed that GmAGL15 was localized in nucleolus and membrane.Pairwise comparison of GmAGL 15 protein with AGL15 revealed a similarity of approximately 69%.An alignment of the genomic and cDNA sequences revealed that GmAGL15 contained eight exons and seven introns,which was similar to AGL15.N-terminal and C-terminal of the soybean sequence displayed overall homology to the AGL15 proteins.Therefore,it is likely that GmAGL15 is the soybean ortholog of AGL15.
In order to study the rule of GmAGL15 gene expression and regulation in soybean,we cloned a 1kb upstream of the translation start site from soybean genome in silico.Promoter sequence analyzed by PLACE showed that it had TATA-box,CA.AT-box and some cis-acting element which regulated specific expression in different organs and responses to stresses,light,and self-feedback.Therefore,GmAGL15 may be regulated by sucrose,auxin and ethylene.Compared the promoter of soybean GmAGL15 with homologue of other plants by FootPrinter analysis,we found that these promoters had conservatism and diversity and the distributing of transcription factor-binding sites had similarity and difference.It implied the accuracy and diversity of GmAGL15 gene expression and regulation.
GmAGL15 was transferred into tobacco by Agrobacterium-mediated method.Real Time RT-PCR assay revealed GmAGL15 expressed in all these lines.But there is no noticeable phenotypic change to the wild-type plants.
In order to investigate the function of GmMADS28,GmMADS28 was transferred to soybean through Agrobacterium-mediated co-transformation system to enhance its expression.62 resistant plantlets were obtained through antibiotic screening,and a few of them were found to be early flowering.RNAi technology has been widely used in resent years.In this paper,Gateway technology was used to replace the conventional method to construct GmMADS28 RNAi vectors.Then the vector was transferred to Agrobacterium C58C1 for its further transformation in plants.
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