辣椒热胁迫相关基因表达分析及功能研究

发布时间：2018-06-09 14:48

  本文选题：辣椒 + 热胁迫　； 参考：《西北农林科技大学》2016年博士论文

【摘要】：辣椒是一种重要的蔬菜作物,因其果实可作为烹饪调味品,含有必需的维生素和其他营养物质而深受人们喜爱。但是,辣椒对高温比较敏感,特别在生殖生长期。一旦遭受持续高温,辣椒的授粉受精会受到严重伤害,从而导致辣椒果实产量和品质的下降。因此,阐明辣椒对热胁迫响应机理有助于寻找提高辣椒耐热性的方法,减轻热胁迫对辣椒造成的伤害。为实现这一目标,首先要对辣椒的热胁迫相关基因进行鉴定分析并研究其功能。本研究鉴定了辣椒的3个热胁迫相关基因家族,包括热激转录因子(Hsf)、热激蛋白70(Hsp70)和热激蛋白20(Hsp20)家族;分析了家族成员的基因结构、保守结构域和系统发育关系;分析了家族成员的组织特异表达和对非生物胁迫的响应;研究了其中3个热胁迫相关基因的功能。主要得到以下结果:1、辣椒CaHsf基因家族共有25个成员,根据保守的氨基酸结构域可分为A、B、C 3个亚家族,其中A亚家族成员最多,B亚家族次之,C亚家族成员最少,只有1个;基因家族成员分布于辣椒12条染色体中的11条,其中11号染色体没有成员分布;家族成员的结构高度保守;基因表达具有组织特异性,至少在根、茎、叶、果皮和胎座中的其中1种组织有表达;家族基因对热胁迫、盐胁迫、渗透胁迫和外源钙离子、腐胺、脱落酸和茉莉酸甲酯有响应。2、辣椒CaHsp20基因家族共有35个成员,结构较为保守,分属于12个亚家族;家族成员分布于辣椒的12条染色体上,其中3、6和8号染色体成员较多,各有5个,2、11和12号染色体成员最少,各有1个;几乎所有家族成员含有内含子数目不超过1个,但是CaHsp27.6例外,有5个内含子;在常温下,家族基因在不同组织中表达水平较低,但受热胁迫强烈诱导;热胁迫处理下,基因在耐热品系R9中的表达峰值出现时间早于热敏品系B6;基因启动子中含有多种逆境响应元件,包括热激元件。3、辣椒CaHsp70基因家族共有21个成员,结构最为保守;根据细胞定位可分为4个亚家族,胞质、内质网、线粒体和质体亚家族,其中胞质亚家族成员最多;家族成员分布于辣椒12条染色体上;基因中内含子数目多样,CaHsp70-2、CaHsp70-13和CaHsp70-20基因中没有内含子,CaHsp70-10中内含子最多,有10个,胞质亚家族成员的内含子数目不超过1个;启动子中含有多种胁迫相关的顺式作用元件,包括热激元件、低温响应元件、防御和胁迫响应元件、植物激素响应元件,其中CaHsp70-10启动子中的热激元件最多;家族基因表达具有组织特异性,同时受热胁迫和Ca2+调控。4、辣椒热激转录因子CaHsfA2基因定位于细胞核,具有转录活性;转基因植株表型和对照相比没有差别,CaHsfA2能够增强转基因植株耐热性和耐盐性,能够调节转基因株系中胁迫相关基因的表达;其启动子有基础活性,受热胁迫、低温和盐胁迫诱导;N-端区域(CaHsfA2△N)虽然缺少激活结构域和核输出信号,但是也能够增强转基因植物耐热性,然而其功能弱于CaHsfA2。5、辣椒热激蛋白CaHsp70-2定位于细胞质和细胞核;CaHsp70-2基因不影响转基因植株的正常生长;CaHsp70-2能够增强转基因植物耐热性,包括本底耐热性、获得耐热性和适度高温耐性,能够降低耐盐性和耐旱性;能调控转基因植株中胁迫相关内源基因表达,包括热激蛋白、热激转录因子、抗坏血酸过氧化物酶和肌醇半乳糖苷合酶基因。6、辣椒热激蛋白CaHsp24.2定位于线粒体和细胞质,也可能定位于叶绿体;转基因植株的生长不受过量表达CaHsp24.2基因的影响;CaHsp24.2能够增强转基因植物耐热性;能够调控转基因植物中热胁迫相关基因表达,其中表达水平变化较为明显的基因有AtHsp17.6B-CI、AtHsa32、AtAPX2和AtHsp90。7、沉默CaHsfA2、CaHsp70-2和CaHsp24.2的辣椒植株表现为株型矮小,叶片畸形;经过高温处理后,和阴性对照相比,沉默植株的耐热性降低;3个基因间存在相互调节的关系,和CaHsp24.2相比,CaHsp70-2对CaHsfA2基因调控更强;CaHsp70-2受CaHsf A2影响较小,受CaHsp24.2影响较大;CaHsp24.2和CaHsp70-2间的相互作用关系要强于与CaHsf A2的作用。
[Abstract]:Capsicum is an important vegetable crop because its fruit is popular as a cooking condiment containing essential vitamins and other nutrients. However, hot pepper is sensitive to high temperature, especially during the reproductive period. Once sustained high temperature, the pollination of chili can be seriously injured, resulting in the production of chili fruit. Therefore, it is necessary to clarify the response mechanism of hot pepper to heat stress and to find a way to improve the heat resistance of chili, and to reduce the damage caused by heat stress. In order to achieve this goal, first of all, we should identify and analyze the related genes of heat stress related genes in chili pepper and study its function. This study identified 3 heat stress related groups in chili pepper. The family, including heat shock transcription factor (Hsf), heat shock protein 70 (Hsp70) and heat shock protein 20 (Hsp20) family, analyzed the gene structure, conserved domain and phylogenetic relationship of family members, analyzed the specific expression of the family members and the response to abiotic stress, and studied the functions of 3 heat stress related genes. The following results are as follows: 1, there are 25 members of the CaHsf gene family, which can be divided into 3 subfamilies of A, B and C according to the conservative amino acid domain. Among them, the A subfamily members are the most, the B subfamily is the most, the C subfamily is the least, only 1; the family members of the gene family are distributed in 11 of the 12 chromosomes of the capsicum, and the 11 chromosome does not have a member distribution. The structure of family members is highly conserved; gene expression is tissue specific, at least 1 tissues are expressed in roots, stems, leaves, pericarp and placenta; family genes have heat stress, salt stress, osmotic stress and exogenous calcium ions, putrescine, abscisic acid and methyl jasmonate have a response to.2, and the CaHsp20 gene family of Capsicum has a total of 35 members. It is more conservative and belongs to 12 subfamilies; family members are distributed on 12 chromosomes of capsicum, of which 3,6 and 8 are more members, each has 5, and the members of the 2,11 and 12 chromosomes are the least, each having 1, but the number of introns is not more than 1, but CaHsp27.6 exceptions, with 5 introns, and family base at normal temperature. The expression level in different tissues is low, but the heat stress is strongly induced. Under the heat stress treatment, the peak expression of gene expression in the heat resistant strain R9 is earlier than that of the thermosensitive strain B6; the gene promoter contains a variety of adverse response elements, including the heat shock element.3, and the 21 members of the chili CaHsp70 gene family are the most conservative. Cell location can be divided into 4 subfamilies, cytoplasm, endoplasmic reticulum, mitochondria and plastid subfamilies, among which the cytoplasmic subfamily members are the most; family members are distributed on 12 chromosomes of capsicum; the number of introns in the gene is diverse, CaHsp70-2, CaHsp70-13 and CaHsp70-20 genes have no introns, and there are 10 introns in CaHsp70-10, and cytoplasmic subfamily. The number of introns of the members of the family is not more than 1; the promoter contains a variety of stress related cis acting elements, including heat shock element, low temperature response element, defense and stress response element, plant hormone response element, among which CaHsp70-10 promoter is the most thermal shock element; family gene expression is tissue specific, and heat stress is also stressed. And Ca2+ regulation of.4, the CaHsfA2 gene of hot chilli heat stimulated transcription factor is located in the nucleus and has a transcriptional activity. The phenotype of the transgenic plant is not different from that of the control. CaHsfA2 can enhance the heat resistance and salt tolerance of the transgenic plants and regulate the expression of the stress related genes in the transgenic lines; the promoter has the basic activity, and the heat stress is low. The N- terminal region (CaHsfA2 Delta N), although lack of activation domain and nuclear output signal, can also enhance the heat tolerance of transgenic plants, but its function is weaker than CaHsfA2.5, and the hot chilli heat shock protein CaHsp70-2 is located in the cytoplasm and nucleus; the CaHsp70-2 base does not affect the normal growth of the transgenic plants; CaHsp70-2 can be used. The heat resistance of transgenic plants was enhanced, including heat resistance and moderate temperature tolerance, salt tolerance and drought resistance, and regulation of stress related endogenous gene expression in transgenic plants, including heat shock protein, heat shock transcription factor, ascorbic acid peroxidant enzyme and inositol galactosidase gene.6, hot chilli hot egg The white CaHsp24.2 is located in the mitochondria and cytoplasm, and may also be located in the chloroplast; the growth of the transgenic plants is not affected by the amount of CaHsp24.2 gene. CaHsp24.2 can enhance the heat resistance of transgenic plants, and can regulate the expression of heat stress related genes in transgenic plants, and the genes with more obvious changes in the expression level in the transgenic plants are AtHsp17.6 B-CI, AtHsa32, AtAPX2 and AtHsp90.7, silent CaHsfA2, CaHsp70-2 and CaHsp24.2 Capsicum plants show short plant type and deformed leaves; after high temperature treatment, the heat resistance of the silent plants is lower than that of negative. The relationship between the 3 genes is regulated by each other, and CaHsp70-2 has a stronger regulation of CaHsfA2 gene compared with CaHsp24.2; CaHsp70-2, CaHsp70-2. The CaHsf A2 is less affected by CaHsp24.2, and the interaction between CaHsp24.2 and CaHsp70-2 is stronger than that of CaHsf A2.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S641.3
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本文编号：2000094