【作者】 黄聪；

【导师】 戴绍军； 刘炜；

【作者基本信息】 哈尔滨师范大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 植物在其生长过程中经常会遭受到不同程度的生物和非生物逆境胁迫,这些胁迫严重影响植物的生长发育,’并对农业生产造成极大的负面影响。研究发现,在长期的生物进化过程中,植物自身已经发展了多种抵抗干旱胁迫的机制,以通过调节自身生理和代谢变化来抵御干旱。其中的一个重要机制就是通过逆境胁迫信号转导来启动逆境胁迫响应基因的表达。在胁迫信号转导途径中,转录因子由于能够调节多个胁迫响应功能基因的表达而备受关注。我们从出口型大花生鲁花14不同发育时期种子混合cDNA文库中克隆鉴定出了3个花生AP2/EREBP类基因,命名为PNDREB1、PNTINY1、PNTINY2以及1个bZIP类基因,命名为PNbZIP1。作为AP2/EREBP家族的一类成员,DREBs类转录因子特异的存在于植物中,参与植物对干旱、高盐和低温等非生物胁迫的应答与响应。而在植物中,bZIP类转录因子参与蛋白与种子贮藏基因表达、光形态发生及器官建成的控制相关。研究PNDREB1、PNTINY1、PNTINY2、PNbZIP1并应用于实践对改良植物的品质,提高植物抗逆性具有很重要的意义。本论文工作取得的主要研究结果如下：1、通过RT-PCR的方法对PNDREB1、PNbZIP1进行了非生物胁迫的表达模式分析,并对PNbZIP1进行组织表达及诱导表达模式分析。对PNDREB1表达模式分析显示,该基因可对低温处理产生响应,低温下表达水平成倍增加；表达水平也受水杨酸诱导而增强,但对乙烯处理响应不明显,且基因的表达受茉莉酸甲脂的抑制。对PNbZIP1表达模式研究显示,其在花生的根、茎、叶、花、种子、果针中均有表达,为组成型表达,且在花和幼小种子中的表达量较高；受低温、乙烯、水杨酸诱导,响应较迅速,但对Nacl、茉莉酸甲脂、ABA响应不明显。PNTINY1能够响应Nacl.低温、乙烯、茉莉酸甲脂、ABA处理条件,并不响应水杨酸。对乙烯响应程度非常强烈。茉莉酸甲脂条件下,响应程度逐渐降低。PNTINY2能够响应Nacl.低温、乙烯、茉莉酸甲脂、ABA、水杨酸处理条件。茉莉酸甲脂条件下,响应程度随着处理时间增加而降低,48小时检则测不到表达。水杨酸条件下,PNTINY2响应程度逐渐增加。2、利用酵母单杂交系统对PNbZIP1的结合及激活活力进行了鉴定,结果显示：PNbZIP1不能与ABRE元件结合,但具有转录激活活性。利用含有DRE顺式元件的酵母单杂交系统验证PNTINY1、PNTINY2的结合及激活活性,结果表明均能够与DRE顺式元件结合并具有转录激活活性。3、T1代PNDREB1转基因烟草经继代后进一步筛选,获得T2代PNDREB1反义转基因烟草共6个株系。构建PNbZIP1的正、反义植物表达载体pCAMBIA1301-PNbZIP1(+)-GUS及pCAMBIA1301-PNbZIP1(-)-GUS,用于烟草转化。目前已获得T2代PNbZIP1正反义转基因材料各10个株系。T1代PNTINY1、PNTINY2转基因烟草经继代后进一步筛选,获得T2代PNTINY1正、反义转基因烟草各6个株系,获得T2代PNTINY2反义转基因烟草2个株系。4、对获得的T2代PNDREB1、PNbZIP1转基因烟草进行非生物胁迫下植株耐逆性分析,发现PNDREB1(-)对低温胁迫较敏感,低温下转基因植株中胁迫相关基因的表达及生理指标的变化都与野生型有所不同,推测该基因在低温信号响应及转导中发挥作用,该基因同时对ABA、Nacl、干旱胁迫也有一定的响应,认为该基因在植物对非生物胁迫综合抗性方面具有一定功能。PNbZIP1耐逆性实验正在进行。本研究为揭示PNDREB1、PNTINY1、PNTINY2、PNbZIP1在非生物逆境下作用及功能,及它们在胁迫信号传导途径的中的调控机制提供了较好的线索,并为进一步研究这两个基因的功能奠定了良好的工作基础。为通过分子育种手段培育综合抗性提高的转基因抗逆新品种提供了理论依据和优良的基因资源。更多还原

【Abstract】 In its growth process of plants are often subjected to different degrees of biotic and abiotic stress, these stress seriously affect plant growth and development, and agricultural production tremendously negative impact. Study found that long-term process of biological evolution; plants have developed their own resistance to drought stress a variety of mechanisms to regulate itself through physiological and metabolic changes in resistance to drought. One important mechanism is through the environmental stress signal transduction to activate gene expression in response to environmental stress. In stress signal transduction, transcription factors regulate several stress response due to the expression of genes of concern.We export large Luhua peanut seed mix of 14 different developmental stages in the cloned cDNA library identified the three peanut AP2/EREBP genes, named PNDREB1, PNTINY1,PNTINY2 and a bZIP genes, named PNbZIP1.As a class AP2/EREBP family members,DREBs specific transcription factor present in plants,involved in plant drought, high salinity and low temperature and other abiotic stress response and response.In plants,bZIP transcription factor involved in seed storage protein gene expression,light organ morphogenesis and control related built.Research PNDREB1,PNTINY1, PNTINY2,PNbZIPl’and applied to practice on improving the quality of plants, improving plant resistance has very important significance.This paper has made the major findings are as follows:1, by RT-PCR method on PNDREBl,PNbZIP1 were non-expression pattern analysis of biological stress, and PNbZIP1 expression and induction of organized expression pattern analysis. Expression pattern of the PNDREB1 analysis revealed that the gene can produce responses to low temperature treatment, low temperature expression doubled; expression levels induced by salicylic acid is enhanced, but not obvious to ethylene response, and gene expression is regulated by jasmonic acid a fat suppression. Expression pattern of the PNbZIPl study shows that in peanut roots, stems, leaves,flowers,seeds,fruit needle was expressed in both the constitutive expression, and in flowers and young seeds in the higher expression; by low temperature, ethylene, salicylic acid, a more rapid response, but Nacl, jasmonic acid methyl ester, ABA response is not obvious. PNTINY1 able to respond Nacl, low temperature, ethylene jasmonic acid methyl ester, ABA treatment conditions, did not respond to salicylic acid. Very strong level of ethylene response. Jasmonic acid methyl ester under the conditions of decreased responsiveness.PNTINY2 able to respond Nacl, low temperature,ethylenejasmonic acid methyl ester,ABA,salicylic acid treatment conditions. Jasmonic acid methyl ester under the conditions of the processing time to respond to the increase with the degree of reduction, while measuring less than 48 hours to check the expression. Acid conditions, PNTINY2 responsiveness gradually increased.2,using yeast one-hybrid system and the activation energy PNbZIP1 binding were identified, the results show:PNbZIP1 cannot ABRE element binding, but the transcriptional activation activity. Containing the DRE cis-element using the yeast one-hybrid system verification PNTINYl,PNTINY2 binding and activation activity, the results show that both can and DRE cis-element binding and transcriptional activation activity.3,Ti transgenic tobacco on behalf of PNDREB1 After further screening by the subculture to obtain T2 generation of PNDREB1 antisense transgenic tobacco plants were six lines. Construction PNbZIP1 of sense and antisense plant expression vector pCAMBIA1301-PNbZIP1(+)-GUS and pCAMBIA1301-PNbZIP1(-)-GUS, used for tobacco transformation. There has been a generation of PNbZIP1 T2 antisense transgenic lines each 10. T1 generation of PNTINY1,PNTINY2 transgenic tobacco by further screening after subculture, were T2 generation of PNTINYl sense and antisense transgenic tobacco lines of all six, were PNTINY2 T2 generation of transgenic tobacco antisense lines 2.4,the obtained T2 generation of PNDREB1,PNbZIPl transgenic tobacco to non-biological stress on plant stress tolerance analysis, we found PNDREB1 (-) to low temperature stress than the sensitive, low temperature, the transgenic plants, stress-related gene expression and physiological changes in both different from the wild type, suggesting that the gene in low-temperature signal transduction in response and the role of the gene while ABA,Nacl,drought stress has a certain response, that the gene in plant resistance to abiotic stress has a comprehensive certain functions.PNbZIPl stress tolerance test in progress.This study reveals PNDREB1,PNTINY1,PNTINY2,PNbZIP1 in non-biological role and function under stress, and they stress signal transduction pathway in the regulation mechanism provides a good clue for the further study the functions of these two genes lay a good working basis. Means of fostering the adoption of integrated molecular breeding to improve resistance to new varieties of genetically modified resilience provides a theoretical basis and excellent genetic resources.更多还原