【作者】 高建杰；

【导师】 姚泉洪；

【作者基本信息】 南京农业大学 ， 果树学， 2011， 博士

【摘要】 长期以来,利用传统的育种手段对多年生木本果树进行遗传改良时,由于受到童期较长等因素的影响,育种进程较为缓慢。基因工程技术的迅猛发展使其成为育种的一种重要的有巨大发展潜力的手段。干旱、盐害、极端温度、化学毒害和氧化胁迫等非生物胁迫严重影响农业生产,并导致环境退化。植物在生长发育过程中,需要对各种逆境信号作出反应,这就要求对各种功能基因的表达进行精确调控。因此,系统研究植物对环境胁迫应答的机制就显得尤为重要。近年来模式植物的研究为果树抗逆的研究提供了难得的机遇。苹果是世界上最重要的果树之一,由于其生态适应性较强,果品营养价值高,耐贮性好,供应周期长,世界上许多国家都将其列为主要消费果品而大力推荐。然而,在果树的栽培过程中,经常会遭遇不同程度的逆境胁迫,而且近年来,全球环境变化加剧,恶劣天气频出,这对苹果的品质和产量都造成很大的影响。在苹果的生产中,为提高其抗逆境胁迫的能力,研究苹果各种抗逆基因迫在眉睫。本文对两个苹果中MYB基因通过拟南芥验证了其功能,以期为利用这些基因进行苹果或其它果树的基因工程改良或表达调控提供理论依据。主要研究结果如下：1.利用PCR方法方法合成了来源于苹果的转录因子MdMYB6。使用Real-Time PCR技术对MdMYB6基因在不同组织以及不同逆境胁迫下的相对转录水平进行了定量分析,结果表明,MdMYB6基因的表达在根中最高,并且受蔗糖、PEG的诱导。2.分析了MdMYB6基因的序列,序列分析表明MdMYB6的开放阅读框(ORF)为939bp,编码一个由312个氨基酸组成的蛋白质。进化树分析推测其基因功能可能与蔗糖响应有关。并利用模式植物拟南芥研究了MdMYB6的功能。在蔗糖浇灌实验中,野生型拟南芥叶片全部变红,而转基因拟南芥则基本没有变化。进一步测定拟南芥花青素含量以及花青素生物合成基因,结果表明,MdMYB6基因能够抑制转基因拟南芥中花青素的生物合成,并且花青素生物合成基因CHS、CHI、DFR、UF3GT、LDOX和FLS也受到MdMYB6基因的抑制,花青素合成调控基因PAP1、PAP2也受到了抑制。同时,我们还又测定了转MdMYB6基因的拟南芥中蔗糖响应和蔗糖运输相关基因,结果表明,MdMYB6改变了蔗糖合成酶基因AtSUS1、蔗糖运输酶AtSUC1、AtSUC2和AtSUC3表达,同时与糖信号相关的UGPase、AtHXK1也发生了改变。3.利用基于PCR的PTDS(PCR-based two-step DNA synthesis,PTDS)方法合成了来源于苹果的转录因子MdMYB10基因并利用模式植物拟南芥研究了这个基因的功能。分析其在山梨醇渗透胁迫条件下的功能表明,在山梨醇胁迫条件下,过量表达MdMYB10基因的拟南芥存活率较野生型显著提高,通过测定一系列生理指标,进一步证明了MdMYB10基因能够在胁迫条件下降低叶绿素的降解,同时提高了类黄酮,脯氨酸的表达,避免了细胞膜的破坏,从而使转基因拟南芥提高了对渗透胁迫的耐受性。随后的RT-PCR证明了在渗透胁迫下,MdMYB10基因能够促进类黄酮合成基因的表达。更多还原

【Abstract】 For a long time, with traditional breeding methods, we face many difficulties such as the slower breeding process caused longer juvenile period while the fruit trees were genetic improving. Genetic engineering technique became one of the importantest potential breeding methods. Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity, and oxidative stress, have enormous damage impact on agriculture and result in the deterioration of the environment. Plants respond and adapt to abiotic stresses by a variety of biochemical and physiological mechanisms, which are associated with the rapid activation of signal transduction pathways. Apple is one of the most important fruit trees in the world. Due to their ecological adaptability, high nutritional value, storability, longer supply cycle, apples are major consumer products and obtain strongly recommended in many countries. However, in the process of planting fruit trees, with different degrees of stress, and in recent years, global environmental have been changed increasingly and then frequent worse weather, which have bad impact on apple’s quality and yield. In order to improve the capacity of its anti-Stress, stress tolerance related genes in a variety of apples is imminent.According to the research hotspot of the model plant Arabidopsis, we studied the expression characterization of these genes, we expect that it will be used in the genetic engineering breeding or regulate gene expression. The main results are as follows:1. Mdmyb6 gene from Malus domestica Borkh. were successfully synthesized by PCR method. The results of Real-Time PCR indicated that the expression of MdMYB6 gene was induced by salt stress, sucrose and PEG, and the transcript level of MdMYB6 gene was the highest in Malus demestic Borkh roots. 2. We analysis the sequence of Mdmyb6 isolated from Malus domestica Borkh. The full-length cDNA of Mdmyb6 has a 939 bp open reading frame (ORF) and encode a MYB transcription factors consisting in 312 amino acids. The Mdmyb6 deduced amino acid sequence showed high identity to sucrose-responsive element binding protein family members. In the sugar watering experiment, almost all of the leaves of WT plants turned red when treated with 180 mM sucrose, while the transgenic line showed no color change. We then assayed anthocyanin accumulation in plants treated with 180 mM sucrose or sorbitol. The transgenic lines showed significantly lower anthocyanin accumulation than the wild type plants. The results of Real Time-PCR indicated that over-expression of MdMYB6 in Arabidopsis influences biosynthesis of anthocyanins, likely by suppressing enzymes involved in anthocyanin biosynthesis such as CHS、CHI、DFR、UF3GT、LDOX and FLS. Over-expression of MdMYB6 also altered the expression of sucrose synthesis (AtSUS1)and transporter genes(AtSUC1、AtSUC2、AtSUC3). At the same time, gene involved in sugar signal gene (UGPase, AtHXKl) also changed.3. Mdmyb10 gene from Malus domestica Borkh were successfully synthesized PCR-based two-step DNA synthesis (PTDS) method.Mdmyb10, a regulatory gene of anthocyanin biosynthesis in apple fruit, into Arabidopsis and analyzed its function to osmotic stress in transgenic plants. Under high osmotic stress, the Mdmyb10 over-expressing plants exhibited growth better than wild-type plants. The elevated tolerance of the transgenic plants to osmotic stress was confirmed by the changes of flavonoids、chlorophyll、malondialdehyde and proline contents. These results preliminarily showed that the MdmyblO can possibly be used to enhance the high osmotic-tolerant ability of plants.更多还原