發(fā)布時(shí)間：2018-06-09 14:48

  本文選題：辣椒 + 熱脅迫��； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：辣椒是一種重要的蔬菜作物,因其果實(shí)可作為烹飪調(diào)味品,含有必需的維生素和其他營(yíng)養(yǎng)物質(zhì)而深受人們喜愛(ài)。但是,辣椒對(duì)高溫比較敏感,特別在生殖生長(zhǎng)期。一旦遭受持續(xù)高溫,辣椒的授粉受精會(huì)受到嚴(yán)重傷害,從而導(dǎo)致辣椒果實(shí)產(chǎn)量和品質(zhì)的下降。因此,闡明辣椒對(duì)熱脅迫響應(yīng)機(jī)理有助于尋找提高辣椒耐熱性的方法,減輕熱脅迫對(duì)辣椒造成的傷害。為實(shí)現(xiàn)這一目標(biāo),首先要對(duì)辣椒的熱脅迫相關(guān)基因進(jìn)行鑒定分析并研究其功能。本研究鑒定了辣椒的3個(gè)熱脅迫相關(guān)基因家族,包括熱激轉(zhuǎn)錄因子(Hsf)、熱激蛋白70(Hsp70)和熱激蛋白20(Hsp20)家族;分析了家族成員的基因結(jié)構(gòu)、保守結(jié)構(gòu)域和系統(tǒng)發(fā)育關(guān)系;分析了家族成員的組織特異表達(dá)和對(duì)非生物脅迫的響應(yīng);研究了其中3個(gè)熱脅迫相關(guān)基因的功能。主要得到以下結(jié)果:1、辣椒CaHsf基因家族共有25個(gè)成員,根據(jù)保守的氨基酸結(jié)構(gòu)域可分為A、B、C 3個(gè)亞家族,其中A亞家族成員最多,B亞家族次之,C亞家族成員最少,只有1個(gè);基因家族成員分布于辣椒12條染色體中的11條,其中11號(hào)染色體沒(méi)有成員分布;家族成員的結(jié)構(gòu)高度保守;基因表達(dá)具有組織特異性,至少在根、莖、葉、果皮和胎座中的其中1種組織有表達(dá);家族基因?qū)崦{迫、鹽脅迫、滲透脅迫和外源鈣離子、腐胺、脫落酸和茉莉酸甲酯有響應(yīng)。2、辣椒CaHsp20基因家族共有35個(gè)成員,結(jié)構(gòu)較為保守,分屬于12個(gè)亞家族;家族成員分布于辣椒的12條染色體上,其中3、6和8號(hào)染色體成員較多,各有5個(gè),2、11和12號(hào)染色體成員最少,各有1個(gè);幾乎所有家族成員含有內(nèi)含子數(shù)目不超過(guò)1個(gè),但是CaHsp27.6例外,有5個(gè)內(nèi)含子;在常溫下,家族基因在不同組織中表達(dá)水平較低,但受熱脅迫強(qiáng)烈誘導(dǎo);熱脅迫處理下,基因在耐熱品系R9中的表達(dá)峰值出現(xiàn)時(shí)間早于熱敏品系B6;基因啟動(dòng)子中含有多種逆境響應(yīng)元件,包括熱激元件。3、辣椒CaHsp70基因家族共有21個(gè)成員,結(jié)構(gòu)最為保守;根據(jù)細(xì)胞定位可分為4個(gè)亞家族,胞質(zhì)、內(nèi)質(zhì)網(wǎng)、線粒體和質(zhì)體亞家族,其中胞質(zhì)亞家族成員最多;家族成員分布于辣椒12條染色體上;基因中內(nèi)含子數(shù)目多樣,CaHsp70-2、CaHsp70-13和CaHsp70-20基因中沒(méi)有內(nèi)含子,CaHsp70-10中內(nèi)含子最多,有10個(gè),胞質(zhì)亞家族成員的內(nèi)含子數(shù)目不超過(guò)1個(gè);啟動(dòng)子中含有多種脅迫相關(guān)的順式作用元件,包括熱激元件、低溫響應(yīng)元件、防御和脅迫響應(yīng)元件、植物激素響應(yīng)元件,其中CaHsp70-10啟動(dòng)子中的熱激元件最多;家族基因表達(dá)具有組織特異性,同時(shí)受熱脅迫和Ca2+調(diào)控。4、辣椒熱激轉(zhuǎn)錄因子CaHsfA2基因定位于細(xì)胞核,具有轉(zhuǎn)錄活性;轉(zhuǎn)基因植株表型和對(duì)照相比沒(méi)有差別,CaHsfA2能夠增強(qiáng)轉(zhuǎn)基因植株耐熱性和耐鹽性,能夠調(diào)節(jié)轉(zhuǎn)基因株系中脅迫相關(guān)基因的表達(dá);其啟動(dòng)子有基礎(chǔ)活性,受熱脅迫、低溫和鹽脅迫誘導(dǎo);N-端區(qū)域(CaHsfA2△N)雖然缺少激活結(jié)構(gòu)域和核輸出信號(hào),但是也能夠增強(qiáng)轉(zhuǎn)基因植物耐熱性,然而其功能弱于CaHsfA2。5、辣椒熱激蛋白CaHsp70-2定位于細(xì)胞質(zhì)和細(xì)胞核;CaHsp70-2基因不影響轉(zhuǎn)基因植株的正常生長(zhǎng);CaHsp70-2能夠增強(qiáng)轉(zhuǎn)基因植物耐熱性,包括本底耐熱性、獲得耐熱性和適度高溫耐性,能夠降低耐鹽性和耐旱性;能調(diào)控轉(zhuǎn)基因植株中脅迫相關(guān)內(nèi)源基因表達(dá),包括熱激蛋白、熱激轉(zhuǎn)錄因子、抗壞血酸過(guò)氧化物酶和肌醇半乳糖苷合酶基因。6、辣椒熱激蛋白CaHsp24.2定位于線粒體和細(xì)胞質(zhì),也可能定位于葉綠體;轉(zhuǎn)基因植株的生長(zhǎng)不受過(guò)量表達(dá)CaHsp24.2基因的影響;CaHsp24.2能夠增強(qiáng)轉(zhuǎn)基因植物耐熱性;能夠調(diào)控轉(zhuǎn)基因植物中熱脅迫相關(guān)基因表達(dá),其中表達(dá)水平變化較為明顯的基因有AtHsp17.6B-CI、AtHsa32、AtAPX2和AtHsp90。7、沉默CaHsfA2、CaHsp70-2和CaHsp24.2的辣椒植株表現(xiàn)為株型矮小,葉片畸形;經(jīng)過(guò)高溫處理后,和陰性對(duì)照相比,沉默植株的耐熱性降低;3個(gè)基因間存在相互調(diào)節(jié)的關(guān)系,和CaHsp24.2相比,CaHsp70-2對(duì)CaHsfA2基因調(diào)控更強(qiáng);CaHsp70-2受CaHsf A2影響較小,受CaHsp24.2影響較大;CaHsp24.2和CaHsp70-2間的相互作用關(guān)系要強(qiáng)于與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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S641.3
，

本文編號(hào)：2000094