本文選題：棉花 + 纖維�。� 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：棉花作為一種重要的經(jīng)濟(jì)作物,其纖維在紡織、民用和工業(yè)原料等方面都有重要的價(jià)值,是一種重要的經(jīng)濟(jì)作物。同時(shí)棉花纖維作為胚珠表皮細(xì)胞突起發(fā)育而成的單細(xì)胞纖維,是研究細(xì)胞分化的很好的材料。眾所周知,纖維突起的數(shù)目和纖維的長度是構(gòu)成棉花產(chǎn)量的重要因素。根據(jù)已有報(bào)道,影響棉花纖維的突起與發(fā)育的因素有很多,如:赤霉素、生長素、乙烯、茉莉酸等植物激素以及MYB類、HD-ZIP類以及其他類型的轉(zhuǎn)錄因子等。本實(shí)驗(yàn)室前期的研究表明高濃度的茉莉酸會抑制纖維的突起,但適量的濃度又能促進(jìn)纖維的伸長。并且JAZ基因在纖維發(fā)育的早期優(yōu)勢表達(dá),說明茉莉酸在纖維發(fā)育過程中具有重要的作用�；谶@些結(jié)果我們進(jìn)行了如下的研究:1.合適濃度的茉莉酸(JA)促進(jìn)纖維的突起為了研究JA對纖維發(fā)育的影響,我們檢測了-3 DPA(Days Post Anthesis)到8 DPA的纖維及胚珠的混合樣中的JA的含量。結(jié)果顯示-1 DPA樣品中的JA含量最高,到0 DPA的時(shí)候有所降低,但都高于其他時(shí)期的樣品中的JA含量。說明JA在纖維發(fā)育早期起關(guān)鍵作用。因此我們檢測了不同突變體(GZnn、GZNn、n2、XinWX和Xu142 fl)和野生型(Xu142和TM-1)材料0 DPA胚珠的JA的含量,發(fā)現(xiàn)在突變體里的JA的含量低于野生型。進(jìn)一步的胚珠培養(yǎng)實(shí)驗(yàn)發(fā)現(xiàn)在0.001μM的條件下,0 DPA的表皮上纖維細(xì)胞的突起數(shù)目比不添加JA的對照及其他濃度的處理有所增加。因此我們認(rèn)為JA對纖維的發(fā)育存在劑量效應(yīng),合適濃度的JA能夠促進(jìn)纖維的突起。2.棉花基因GhJAZ2的克隆和序列分析根據(jù)NCBI的序列信息,我們克隆了JA信號傳導(dǎo)路徑上的關(guān)鍵基因JAZ(ES802380)。其編碼的蛋白質(zhì)包含JAZ家族高度保守的TIFY結(jié)構(gòu)域和C-端的Jas結(jié)構(gòu)域。將ES802380與擬南芥的JAZ家族的蛋白進(jìn)行比對發(fā)現(xiàn),其與AtJAZ1與AtJAZ2的蛋白最同源,其中與AtJAZ1相似度為33.21%,與AtJAZ2相似度為43.28%,因此將該基因命名為GhJAZ2。基因組織表達(dá)分析發(fā)現(xiàn)GhJAZ2在根、下胚軸、花器官和-1 DPA的胚珠里高量表達(dá)。3.在棉花中超表達(dá)GhJAZ2基因抑制纖維長絨和短絨的突起,以及長絨的伸長。為了驗(yàn)證GhJAZ2基因在棉花發(fā)育過程中發(fā)揮的功能,我們構(gòu)建了CaMV35S超表達(dá)載體(CaMV 35S::GhJAZ2)和干涉載體(CaMV 35S::iGhJAZ2)并轉(zhuǎn)化棉花。多年的重復(fù)試驗(yàn)結(jié)果發(fā)現(xiàn),不同超表達(dá)株系的纖維長度均比對照組變短,種子變小。掃描電鏡的結(jié)果及統(tǒng)計(jì)數(shù)據(jù)顯示超表達(dá)株系長絨和短絨突起的數(shù)目比對照少。綜上所述,在棉花中超表達(dá)GhJAZ2引起長絨短絨的突起都變少,纖維變短,種子變小,但是干涉GhJAZ2無明顯表型。4.GhJAZ2通過與GhMYB25-like、GhGL1、GhMYC2、GhWD40和GhJI1等互作抑制纖維的突起為了進(jìn)一步研究GhJAZ2影響纖維發(fā)育的作用機(jī)理,利用酵母雙雜技術(shù)以GhJAZ2作為誘餌蛋白篩選-2 DPA~4 DPA的酵母雙雜文庫,獲得了將近80個(gè)陽性克隆。最終我們關(guān)注了其中的5個(gè)互作蛋白,它們分別是R2R3-MYB類轉(zhuǎn)錄因子GhMYB25-like和GhGL1、bHLH類轉(zhuǎn)錄因子GhMYC2、WD40重復(fù)蛋白家族GhWD40和一個(gè)未知功能蛋白GhJI1。并且運(yùn)用BIFC的方法證實(shí)了GhJAZ2與這5個(gè)轉(zhuǎn)錄因子確實(shí)存在互作。5.GhJAZ2通過與GhMYB25-like互作抑制突起目前,BIFC驗(yàn)證的這5個(gè)互作蛋白中只有GhMYB25-like被證實(shí)參與纖維的突起,其他幾個(gè)均沒有相關(guān)文獻(xiàn)報(bào)道。報(bào)道表明,降低GhMYB25-like的表達(dá)量之后會導(dǎo)致纖維的突起減少,甚至光籽的表型。同時(shí)我們對棉花CaMV35S::GhMYB25-like超表達(dá)株系的表型鑒定結(jié)果表明,提高GhMYB25-like表達(dá)量會使0 DPA胚珠的纖維突起的數(shù)目增加。因此,我們將GhJAZ2的超表達(dá)株系與GhMYB25-like超表達(dá)株系進(jìn)行雜交,發(fā)現(xiàn)雜交F1代(GhJAZ2×GhMYB25-like)的0 DPA胚珠表面纖維突起的數(shù)目與GhJAZ2的超表達(dá)株系的突起數(shù)目沒有差異。同時(shí)在煙草的原生質(zhì)體瞬時(shí)轉(zhuǎn)化體系中,驗(yàn)證了GhMYB25-like存在轉(zhuǎn)錄激活功能,并且在GhJAZ2存在的時(shí)候GhMYB25-like的轉(zhuǎn)錄激活功能受到了抑制。因此,我們認(rèn)為GhJAZ2通過與棉花纖維起始關(guān)鍵基因GhMYB25-like互作并抑制了其轉(zhuǎn)錄激活功能,從而抑制了纖維的突起。6.棉花JAZ家族中存在功能冗余在GhJAZ2的RNAi株系中,長絨短絨的數(shù)目以及長絨長度均與野生型沒有差異。為了解釋該表型,我們參考已經(jīng)測序的陸地棉基因組數(shù)據(jù),共獲得棉花JAZ家族的30個(gè)成員。隨后通過基因表達(dá)熱圖分析發(fā)現(xiàn)其中3個(gè)基因(Gh_D07G0152,Gh_D10G0531和Gh_D05G0379)的表達(dá)模式與GhJAZ2相似,并且FPKM值是大于GhJAZ2的。進(jìn)一步的進(jìn)化樹分析發(fā)現(xiàn)另外2個(gè)基因(Gh_D08G2564和Gh_D05G0352)與GhJAZ2最同源。JAZ家族基因在陸地棉TM-1的不同組織中的表達(dá)模式分析發(fā)現(xiàn)其中Gh_D01G1406在-1 DPA中有一個(gè)表達(dá)高峰。將上述的6個(gè)基因的全長分別克隆出來用酵母雙雜的方法驗(yàn)證與GhMYB25-like、GhGL1、GhMYC2、GhWD40和GhJI1之間是否存在互作,結(jié)果表明只有Gh_D01G1406能夠與這5個(gè)蛋白分別互作,其他的幾個(gè)均不與這5個(gè)蛋白存在互作。并且我們檢測了該基因在GhJAZ2的轉(zhuǎn)基因株系中的表達(dá)量,發(fā)現(xiàn)在干涉株系中該基因的表達(dá)量有所上調(diào)。據(jù)此,我們認(rèn)為棉花干涉的株系沒有出現(xiàn)表型可能是由于JAZ家族中存在與GhJAZ2功能冗余的其他成員。
[Abstract]:As an important economic crop, cotton is an important economic crop in textile, civil and industrial materials. It is an important economic crop. At the same time, cotton fiber is a single cell fiber developed from the protuberance of the ovule epidermal cells. It is a very good material to study the differentiation of cells. Fiber length is an important factor in the production of cotton. According to the reports, there are many factors affecting the protuberance and development of cotton fiber, such as gibberellin, auxin, ethylene, jasmonic acid and other plant hormones as well as MYB, HD-ZIP and other types of transcription factors. The protuberance of the fiber was inhibited, but a proper concentration could promote the elongation of the fiber. And the expression of JAZ gene at the early stage of fiber development showed that jasmonate played an important role in the development of fiber. Based on these results, we have studied the following results: 1. the appropriate concentration of jasmonic acid (JA) promotes the protruding of fiber in order to study the JA pair The effect of fiber development, we detected the content of JA in the mixed samples of -3 DPA (Days Post Anthesis) to 8 DPA fibers and ovules. The results showed that JA content in -1 DPA samples was the highest, and decreased at the time of 0 DPA, but higher than the JA content in the samples of other periods. The contents of JA of 0 DPA ovules of different mutants (GZnn, GZNn, N2, XinWX and Xu142 FL) and wild type (Xu142 and TM-1) materials were detected. The JA content in the mutant was found to be lower than that in the wild type. Further ovule culture experiment found that the number of fibrous cells on the surface of the surface of the 0 epidermis was compared with that of the 0.001 micron M. The treatment of his concentration has increased. Therefore, we think JA has a dose effect on the development of fiber. A suitable concentration of JA can promote the cloning and sequence analysis of the.2. cotton gene GhJAZ2 of the fiber. According to the sequence information of NCBI, we cloned the key gene JAZ (ES802380) on the JA signal transduction pathway. The encoded protein contains JA. The highly conserved TIFY domain of the Z family and the Jas domain of the C- end. Compared the protein of ES802380 to the Arabidopsis JAZ family, it was found that it was the most homologous to the proteins of AtJAZ1 and AtJAZ2, in which the similarity between AtJAZ1 and AtJAZ2 was 33.21% and the AtJAZ2 similarity was 43.28%. Therefore, the gene was named as the GhJAZ2. gene tissue expression analysis to find GhJAZ2 in The high expression of.3. in the ovule of the root, hypocotyl, flower organ and -1 DPA in the ovule of the cotton was overexpressed by the GhJAZ2 gene to inhibit the protuberance of fiber long velvet and short velvet, as well as the elongation of long velvet. In order to verify the function of the GhJAZ2 gene in the development of cotton, we constructed the CaMV35S overexpression vector (CaMV 35S:: GhJAZ2) and the interference carrier (CaMV 35S:: iGh). JAZ2) and transformation of cotton. Many years of repeated test results showed that the fiber length of different hyper expression lines became shorter and the seeds were smaller than those of the control group. The results of scanning electron microscopy and statistics showed that the number of overexpressed lines and short fleece projections was less than that of the control. In summary, the overexpression of GhJAZ2 in cotton flowers caused the changes of the short velvet. Less fiber, shorter fibers and smaller seeds, but the interference GhJAZ2 has no obvious phenotypic.4.GhJAZ2 through the interaction with GhMYB25-like, GhGL1, GhMYC2, GhWD40 and GhJI1 to inhibit the fiber development in order to further study the mechanism of GhJAZ2 affecting fiber development, using yeast double heterozygosity to select GhJAZ2 as bait protein for screening -2 DPA~4 DPA. Nearly 80 positive clones were obtained. Finally, we paid attention to 5 interactivity proteins, which are R2R3-MYB class transcription factors GhMYB25-like and GhGL1, bHLH transcription factor GhMYC2, WD40 repeat protein family GhWD40, and an unknown functional protein GhJI1. and confirmed that GhJAZ2 and these 5 transcription factors are true. The existence of interaction.5.GhJAZ2 by interacting with GhMYB25-like and inhibiting protuberance at the present time, only GhMYB25-like in the 5 interacted proteins verified by BIFC has been confirmed to be involved in the protuberance of the fiber, and the other few are not reported in the relevant literature. The results of phenotypic identification of cotton CaMV35S:: GhMYB25-like overexpressed strain showed that increasing GhMYB25-like expression could increase the number of fibrous protrusions in 0 DPA ovules. Therefore, we hybridize the overexpression strain of GhJAZ2 with the GhMYB25-like overexpression strain, and found the 0 DPA ovule surface fiber of the F1 generation (GhJAZ2 * GhMYB25-like). The number of vascular protrusions is not different from the number of GhJAZ2 overexpressed strains. Meanwhile, in the transient transformation system of protoplasts of tobacco, GhMYB25-like has a transcriptional activation function and the transcriptional activation function of GhMYB25-like is suppressed when GhJAZ2 exists. Therefore, we believe that GhJAZ2 is used to play with cotton fibers. The key gene GhMYB25-like interacts and inhibits its transcriptional activation function, which inhibits the fibrous protuberance of the.6. cotton JAZ family with functional redundancy in the RNAi line of the GhJAZ2. The number of long velvet and the length of the long velvet are not different from that of the wild type. In order to explain the phenotype, we refer to the genome number of the land cotton that has been sequenced. A total of 30 members of the cotton JAZ family were obtained. Subsequently, the expression pattern of 3 genes (Gh_D07G0152, Gh_D10G0531 and Gh_D05G0379) was found to be similar to GhJAZ2, and the FPKM value was greater than GhJAZ2. Further evolutionary tree analysis found that the other 2 genes (Gh_D08G2564 and Gh_D05G0352) were the most homologous to GhJAZ2. The expression pattern analysis of AZ family gene in different tissues of upland cotton TM-1 found that Gh_D01G1406 had an expression peak in -1 DPA. The whole length of the 6 genes was cloned separately to verify the interaction between GhMYB25-like, GhGL1, GhMYC2, GhWD40 and GhJI1 with yeast double heterozygosity. 406 can interact with these 5 proteins separately, and the others do not interact with the 5 proteins. And we detected the expression of the gene in the GhJAZ2 transgenic line and found that the expression of the gene was up in the interference strain. Accordingly, we think that the phenotype of the cotton stem related strains may not be due to the JAZ family. There are other members in the family with the redundancy of the GhJAZ2 function.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S562

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本文編號：2099896