摘要
DREB(干旱响应元件结合蛋白)转录因子是近十年来新发现的在植物生长发育、激素调节和抵抗逆境等方面发挥重要作用的植物特异性转录因子。在逆境胁迫下,它通过与DRE元件(脱水应答元件)特异性结合,从而激活许多逆境诱导基因的表达,提高植物对逆境的抵抗和忍耐力,使植物的抗逆性得到综合性改良,是迄今为止最理想的植物抗逆工程基因。
本研究通过同源克隆的方法从海岛棉中克隆了一个DREB转录因子基因,然后将其转入烟草,对它的基本特性进行了研究,证实了这个基因具有DREB类转录因子的基本特性,它在转基因烟草中的过量表达可以提高烟草抵抗非生物胁迫的能力。主要研究结果如下:
1.根据已发表的陆地棉GhDREB1基因序列设计特异性引物P1和P2,进行RT-PCR扩增,得到一个长651bp,编码216个氨基酸的基因,命名为XHDREB。同源性分析表明,XHDREB与陆地GhDREB1,拟南芥CBF1/DREB1B和DREB1A/CBF3在氨基酸水平上的序列相同性分别为85.19%,47.32%和46.64%。
2.分析XHDREB基因编码的氨基酸序列,其相对分子量为24.6 kDa,等电点为9.46,它具备DREB1转录因子的特征:含有58个氨基酸组成的AP2 DNA结合域以及两个DREB1/CBF特征氨基酸序列PKKRAGRKKFRETR和DSAWR。
3.设计含有KpnⅠ和PstⅠ酶切位点的特异性引物DR-1和DR-2,用KpnⅠ和PstⅠ分别对XHDREB基因和pCAMBIA2300-35SOCS质粒进行双酶切,用T4 DNA Ligase连接,构建植物表达载体pC-XHDREB。
4.利用农杆菌介导法转化烟草,通过Kan抗性、PCR及RT-PCR筛选鉴定阳性烟草。对转基因烟草进行干旱、高盐等抗逆性分析并且对其相关生理生化指标进行测定,结果显示转基因烟草对这些非生物胁迫均有一定的抵抗能力。
3.利用荧光定量RT-PCR对XHDREB在转基因烟草中的表达情况进行分析。结果显示:XHDREB在干旱胁迫下的表达情况与陆地棉GhDREB1的表达情况有所不同,但与拟南芥以及其他植物中DREB1/CBF类基因的相似,在高盐胁迫下的表达情况与陆地棉GhDREB1、拟南芥以及其他植物中DREB1/CBF类基因的相似。但是在低温胁迫下的表达情况与陆地棉GhDREB1、拟南芥以及其他植物中DREB1/CBF类基因有一定差异。
DREB(dehydration responsive element binding protein)transcription factors are a family of functionally diverse proteins.They are unique to plants and play an important role in regulation of plant growth and development,homone reguation and responses to various stresses.The DREB transcription factor can specifically combine with the DRE(dehydration responsion element)and activate expression of many stress-induced genes under the stress and increasing the capacity of resistance and endurance to adversity of plant,is the best adversity resistance gene in plant by far.
In this study,homology clonging methods in the new cloned from island cotton,a DREB transcription factor genes into tobacco by its,its basic characteristics and biological functions have been studied and proved than genes DREBs of transcription factors basic characteristics,which in transgenic tobacco plants over-expression can improve the capacity of resistance to abiotic stress.The major findings are as follows:
1.According to published gene sequence of land cotton GhDREB1 transcription factor,specific primers P1 and P2 were designed and a gene with 651bp in length were amplified by RT-PCR.The gene contained a complete sequence encoding 216 amino acids,named XHDREB.Homological analysis showed that 85.19% amino acids in the sequences were similar to cotton GhDREB1 gene,47.32% amino acids in the sequences were similar to AtCBF1/DREB1B and 46.64% amino.acids in the sequences were similar to AtDREB1A/CBF3.
2.Analysis indicated that the deduced relative molecular weight of the amino acid was 24.6 kDa for XHDREB.It contains the typical characteristics to DREB1 transcription factor:58 amino acids constitute a DNA binding region AP2 and two typical characteristics amino acids sequence to DREB1/CBF:PKKRAGRKKFRETR and DSAWR.
3.The sensed specific primer DR-1 and DR-2,containing KpnⅠand PstⅠrestriction sites,were designed.Plasmid containing XHDREB gene and pCAMBIA2300-35SOCS vector was digested with KpnⅠand PstⅠ,respectively.The T4DNA ligase was used to be ligated,sensed plant expression vector pC-XHDREB was built.
4.Transforming tobacco by using agrobacterium-mediated.Screening and identification of positive tobacco by Kan resistance,PCR and RT-PCR.The drought,high salt resistance and other resistance of transgenic tobacco were analysised.And the biochemical and physiology quota were measured,the results indicate that the transgenic tobacco had some of resistance ability to these abiotic stress.
5.The expression of XHDREB in transgenic tobacco were analyzed by using real time quantitative RT-PCR.There is difference on expression levels of XHDREB under drought stress between upland GhDREB1 and island cotton,But the expression levels of XHDREB in island cotton is similar to the DREB1/CBF genes of arabidopsis and other plant.The expression levels of XHDREB under high salt stress in island cotton is similar to upland GhDREB1,arabidopsis and other plants.But the expression levels of XHDREB under cold stress in island cotton is different from upland GhDREB1,arabidopsis and other plant.
引文
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