【作者】 张勇；

【导师】 汤浩茹；

【作者基本信息】 四川农业大学 ， 果树学， 2009， 博士

【摘要】 草莓是目前果树保护地生产中种植面积最大的一个树种,由于南方冬季温度较高,草莓的越冬保护措施不严格,再加上温室的低温逆转现象,草莓在栽培过程中往往会受到偶发性的短时低温伤害,从而给草莓生产带来巨大损失。利用基因组学和分子生物学如克隆、基因操作、遗传转化等技术可以克服传统育种手段的不足,可以为研究草莓耐寒机制和抗寒育种提供新的思路和方法。CBF(CRT/DRE bindingfactor,CBF)是一类与低温胁迫相关的转录因子,它能够特异地结合到含有CRT/DRE元件的COR基因启动子区,从而激活COR基因的表达进而提高植物的抗寒性。因此,CBF类转录因子能够综合地改良植物的抗寒性状,是目前比较理想的植物抗逆工程基因。本试验以草莓栽培品种‘丰香’(Fragaria×ananassa cv.Toyonaka)为试材,通过与GenBank上发表的几种植物CBF基因进行同源比较,根据氨基酸序列保守区设计简并引物,采用热启动降落PCR方法扩增得到草莓转录因子Facbf1基因的中间片段。在此基础上,通过SON-PCR和RT-PCR,从草莓中分离克隆了Facbf1基因编码区全长cDNA序列,GenBank登录号为FJ767754。该片段长794 bp,包含一个长为636bp的完整ORF,编码1个由211个氨基酸残基组成的蛋白质,预测分子量为23.4 kDa,等电点为6.53。氨基酸序列分析表明FaCBF1蛋白含有一个保守的AP2结合域,在AP2结合域的两端含有两段保守的短多肽序列PKK/RPAGRxKFxETRHP和DSAWRL。利用半定量RT-PCR对Facbf1基因进行差异表达研究,结果表明Facbf1属于冷诱导差异表达基因。未经冷诱导处理的草莓幼叶内Facbf1基因不表达;经冷处理后,Facbf1在短时间内转录水平迅速增加,在12 h时表达量达到高峰并可以维持到24 h,而在冷胁迫的后期其表达水平开始下降。分析FaCBF1蛋白的理化特性,结果表明FaCBF1属易溶、亲水性强的蛋白。系统进化树分析表明FaCBF1蛋白属于CBF基因家族,与同属植物具有比较近的进化关系。二级结构预测结果显示,FaCBF1蛋白主要以α-螺旋和无规则卷曲两种形式出现。采用EsyPred3D同源模构建的三维模型显示,FaCBF1蛋白三维结构在N端形成一个α-螺旋,C端形成3个β-折叠,并与GCC-box结合蛋白有着结构上的相似性。采用网络服务器对FaCBF1的功能进行了预测,结果表明FaCBF1属于CBF家族的一员,含有保守的AP2结合域,具有多个磷酸化位点和O-糖基化位点,不含信号肽和跨膜结构。另外,对CBF的电子表达谱分析表明,CBF在植物中几乎整株表达,而且在多种组织和不同发育阶段均能表达,这也充分说明了CBF在植物生长发育巾具有重要地位。冷驯化在轻至中等的低温胁迫时有效,如果温度过低或时间过长还会导致植物死亡。草莓幼苗经0℃低温锻炼后,各种生理指标的测试表明,草莓幼苗对低温环境具有一定的冷适应能力,即在一定程度上提高了植株的抗寒性。因此,适当的低温处理可以在一定程度上诱导草莓抗寒力的提高。这可能是外界温度下降时,植物感受低温信号,并传递低温信号到核内激活转录因子CBF,进而调节基因转录或引起一系列生理生化反应,如保护酶(SOD、CAT、APX和GR等)活性提高,渗透调节物质(游离脯氨酸、可溶性蛋白和可溶性糖)含量增加以及光合色素(叶绿素和类胡萝卜素)含量下降,并且草莓Facbf1基因的表达与这些物质的变化密切相关。由此可认为植物在低温锻炼过程中往往会发生生理和代谢上的改变从而适应低温,是一个活跃的生理生化过程,植物抗寒力的提高是低温诱导所引起的转录调控因子CBF表达水平以及一系列细胞结构和各种生理生化代谢适应性变化的综合结果。上述研究对于填补园艺植物抗寒性生理及分子生物学研究具有重要的理论意义,并可对今后其他农作物及园艺植物抗寒性的遗传改良奠定了重要的基础,具有一定的应用价值和现实意义。更多还原

【Abstract】 Strawberry production is the biggest planting area at present.The temperature is warm in south,the overwintering safeguard of strawberry is not severe and the low temperature reversion phenomena occurred so that strawberry would suffer little low temperature injury contingently during planting and great loss was peoduced.CBF(CRT/DRE binding factor, CBF) is a type of transcription factors related to abiotic stresses.These proteins are able to discern and bind the CRT/DRE element in the promoter of COR gene concerned with cold, resulting in enhancing the cold-resistance of plants.Therefore,it would be a good strategy to improve tolerance of various kinds of agriculturally important plants to abiotic stresses by CBF genes transfer.In this present studies,a pair of degenerate primers which was designed by comparing CBF genes from GenBank,and the conserved region with a length of 464 bp of the transcription factor CBF gene by touch down PCR from Fragaria×ananassa.Then,a full length of open reading frame of CBF gene was obtained by SON-PCR and RT-PCR.The cDNA was 794 bp in length and a real CBF gene of strawberry designed as Facbfl with an accession number FJ767754 in GenBank.The cloned cDNA contains an ORF of 636 bp, which encodes for a polypeptide of 211 amino acid residues with a molecular mass of 23.4 kDa and a pI of 6.53.FaCBF1 sequence contains a functional domain:AP2 binding domain, on the both side of which there exit two short peptides:PKK/RPAGRxKFxETRHP and DSAWRL.Semi-quantitative RT-PCR is used to analysis the expression of Facbfl gene under different time of treatment.The results showed that without a cold treatment there was no expression of Facbfl genes.But after the cold treatment,the level of Facbfl transcription was rapid increased in a short period of time and reached a peak at 12h and may last up to 24h and then declined.Protein structure was predicted with different methods.The results of primary structure analysis showed that FaCBF1 was soluble and hydrophobic and belonged to CBF family.Secondary structure analysis indicated that FaCBF1 contains conserved Alpha helix and random coils domain.3D structure of protein coding by the gene was predicted using the ESyPred3D software.It contains oneα-helices and threeβ-sheets structural domain and looks like the GCC-box binding protein.Function prediction was carried out in net server. The results showed that FaCBF1 was a member of CBF family,containing AP2-binding domain with some phyosphorylation and O-glycosylation sites,and it had no signal and trasmembrane domain.Electronic expression profiles revealed that FaCBF1 expressed in whole plants and during different development,and involved in different abiotic stresses. All of the results suggest that FaCBF1 played an important role in the growth a development of plants.Cold acclimation can enhance the ability of cold-resistance,but if the temperature is too low or too long it will cause the death of plants.Under different times of lowtemperature treatment,the target of a variety of physiological tests showed that the lowtemperature environment of strawberry seedlings must have the ability to adapt to the cold, that is,to some extent,improve the plant’s cold-resistance,deal with one of 0℃can significantly improve plant adaptation to low temperature.Therefore,an appropriate lowtemperature treatment can be induced to enhance the cold-resistance of strawberry.This may be when the outside temperatures drop,plants feel low-temperature signals,and transmission of low-temperature signals to the nucleus to activate transcription factors, thereby regulating gene transcription or give rise to a series of physiological and biochemical reactions,such as antioxidant enzymes(SOD,CAT,APX and GR) activities and increased the contents of proline,soluble protein and soluble sugar,as well as chlorophyll and carotenoid content decreased,the adaptive changes of these substances laid the foundation of enhancing the cold-resistance of plants.It can be seen that lowtemperature induced the physiological and metabolic changes of plants in order to adapt to low temperature.This is an active physiological and biochemical process,which improve the cold-resistance of plants.These obtained results and conclusions provided important information not only for the physiology and molecular biological research of cold-resistance in horticulture plants but also for the genetic improvement of cold-resistance in agriculture and horticulture plants.更多还原