兩個百合雄性不育相關(guān)基因的克隆和功能初步分析
發(fā)布時間：2018-10-19 09:50
【摘要】：百合一直以其花姿優(yōu)雅,氣度不凡具有極高的觀賞價值而著稱。其在顏色,圖案和香味方面的吸引力而被顧客高度重視,在世界范圍的切花行業(yè)中占有極高的市場份額。然而它具有巨大的花藥,同時伴有大量的花粉,如果花藥不能及時去除,則花粉會污染花瓣和周圍表面,沾染衣物甚至?xí)䦟?dǎo)致人類對花粉過敏等癥狀。因為消費者不愿購買有污染的百合花,且人工摘除花藥又費時費力。因此,控制花藥的發(fā)育和減少花粉量的生產(chǎn),同時又不影響百合花的美觀一直是百合的分子育種的焦點。本實驗選取不產(chǎn)花粉的"無粉白"百合雄蕊和同一時期有粉百合雄蕊為實驗材料,結(jié)合之前轉(zhuǎn)錄組分析數(shù)據(jù)通過熒光定量篩選鑒定基因,RT-PCR克隆得到C2基因的ORF序列,隨即克隆其5'端序列,并在其啟動子序列中找到與花藥特異性相關(guān)的表達元件。還通過同源克隆的方法克隆得到C4基因,該基因與控制雄性不育基因MMD1具有較高的同源性,并對該基因進行一系列的生物信息學(xué)分析。具體研究結(jié)果如下:1.通過熒光定量PCR分析鑒定,發(fā)現(xiàn)C2基因在無粉百合雄蕊中的表達明顯遲于其在有粉百合雄蕊中的表達,且表達量很低。結(jié)合其在百合不同器官中表達量分析,C2基因在花藥中表達最高。這說明C2基因主要的作用位點在花藥中。2.在有粉可育雄蕊與無粉不可育雄蕊中分別通過RT-PCR克隆到C2基因ORF區(qū),全長為774 bp,通過序列比對分析發(fā)現(xiàn)其ORF區(qū)沒有突變,推測可能其發(fā)揮作用的區(qū)域在啟動子部分。3.在有粉可育雄蕊與無粉不可育雄蕊中分別通過染色體步移的方法克隆其啟動子序列,且有粉可育材料中克隆得到序列619 bp,無粉不可育雄蕊中克隆得到片段395 bp。通過啟動子分析軟件NewPlace預(yù)測發(fā)現(xiàn)啟動子序列中含有TATA-box以及CAAT-box,其中TATA-box分別位于轉(zhuǎn)錄起始位點"A"位于起始密碼子上游-53 bp和-54 bp處,CAAT-box位于起始密碼子上游-132 bp和-167 bp處。在該序列中還含有大量的順式作用元件,如花特異性表達元件如:GTGA、AGAAA和TGTGG及其它順勢作用元件。4.通過同源克隆方法得到C4基因,結(jié)合轉(zhuǎn)錄組數(shù)據(jù)分析發(fā)現(xiàn),該基因與已報道的控制雄性不育基因PHD-鋅指蛋白白 MMD1基因具有較高的同源性,經(jīng)過一系列的生物信息學(xué)分析發(fā)現(xiàn),C4基因與鳳仙花MMD1同源性最高為57%,該基因氨基酸序列不存在跨膜區(qū)域,且不存在信號肽,亞細胞定位推測其在細胞核上,其結(jié)構(gòu)域含有PHD-鋅指蛋白結(jié)構(gòu)域,且所有的活性位點都位于這個區(qū)域,具有鋅指蛋白酶的活性,與之前預(yù)測的相符,為后續(xù)研究此基因奠定基礎(chǔ)。
[Abstract]:Lily has always been famous for its elegant flower posture, extraordinary demeanor with extremely high ornamental value. Its appeal in color, design and fragrance is highly valued by customers and has a high market share in the world-wide cut-flower industry. However, it has a huge anther, accompanied by a large number of pollen, if the anther can not be removed in time, the pollen will contaminate the petals and the surrounding surface, and even cause human allergies to pollen symptoms. Because consumers do not want to buy contaminated lilies, and manual extraction of anthers is time-consuming and laborious. Therefore, controlling anther development and reducing pollen production without affecting the beauty of lilies has always been the focus of molecular breeding of lily. In this experiment, the pollen free "powdery white" lily stamen and the pollen lily stamen at the same time were selected as the experimental materials. The ORF sequence of C2 gene was cloned by RT-PCR by fluorescence quantitative screening and identification according to the previous transcriptome analysis data. The 5 '-terminal sequence was cloned and the anther specific expression elements were found in the promoter sequence. The C4 gene was cloned by homologous cloning, which had high homology with the male sterility control gene MMD1, and was analyzed by a series of bioinformatics. The results are as follows: 1. Fluorescence quantitative PCR analysis showed that the expression of C2 gene in the stamen of non-pollen lily was significantly later than that in the stamen of the pollen lily, and the expression of C2 gene was very low. Combined with the analysis of the expression of C2 gene in different organs of lily, C2 gene expression was the highest in anthers. This indicates that C2 gene is mainly located in anthers. 2. The C2 gene ORF region was cloned from the sterile and sterile stamens by RT-PCR. The total length of the C2 gene was 774 bp,. No mutation in the ORF region was found by sequence alignment analysis. It was speculated that the region that might play a role was in the promoter part. 3. The promoter sequence was cloned by chromosome step method in the sterile and sterile stamens, and the sequence 619 bp, was cloned from the pollen fertile material. The fragment 395 bp. was cloned from the sterile stamen and the sterile unfertile stamen. Promoter analysis software NewPlace predicted that the promoter sequence contained TATA-box and CAAT-box, where TATA-box was located at -53 bp and -54 bp upstream of the initiation codon, and CAAT-box was located at -132 bp and -167 bp upstream of the start codon, respectively. The sequence also contains a large number of cis-acting elements, such as floral specific expression elements such as: GTGA,AGAAA and TGTGG and other homeotropic elements. 4. C4 gene was obtained by homologous cloning method. Combined with transcriptome data analysis, it was found that this gene had high homology with PHD- zinc finger protein white MMD1 gene, which controlled male sterility gene. A series of bioinformatics analysis showed that the highest homology of C4 gene and MMD1 was 57. The amino acid sequence of C4 gene had no transmembrane region and no signal peptide. The subcellular localization suggested that C4 gene was located in the nucleus. The domain contains PHD- zinc finger protein domain, and all the active sites are located in this region, which has the activity of zinc finger protease, which is consistent with the previous prediction, which lays the foundation for further study of this gene.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S682.29;Q943.2
，
本文編號：2280775
[Abstract]:Lily has always been famous for its elegant flower posture, extraordinary demeanor with extremely high ornamental value. Its appeal in color, design and fragrance is highly valued by customers and has a high market share in the world-wide cut-flower industry. However, it has a huge anther, accompanied by a large number of pollen, if the anther can not be removed in time, the pollen will contaminate the petals and the surrounding surface, and even cause human allergies to pollen symptoms. Because consumers do not want to buy contaminated lilies, and manual extraction of anthers is time-consuming and laborious. Therefore, controlling anther development and reducing pollen production without affecting the beauty of lilies has always been the focus of molecular breeding of lily. In this experiment, the pollen free "powdery white" lily stamen and the pollen lily stamen at the same time were selected as the experimental materials. The ORF sequence of C2 gene was cloned by RT-PCR by fluorescence quantitative screening and identification according to the previous transcriptome analysis data. The 5 '-terminal sequence was cloned and the anther specific expression elements were found in the promoter sequence. The C4 gene was cloned by homologous cloning, which had high homology with the male sterility control gene MMD1, and was analyzed by a series of bioinformatics. The results are as follows: 1. Fluorescence quantitative PCR analysis showed that the expression of C2 gene in the stamen of non-pollen lily was significantly later than that in the stamen of the pollen lily, and the expression of C2 gene was very low. Combined with the analysis of the expression of C2 gene in different organs of lily, C2 gene expression was the highest in anthers. This indicates that C2 gene is mainly located in anthers. 2. The C2 gene ORF region was cloned from the sterile and sterile stamens by RT-PCR. The total length of the C2 gene was 774 bp,. No mutation in the ORF region was found by sequence alignment analysis. It was speculated that the region that might play a role was in the promoter part. 3. The promoter sequence was cloned by chromosome step method in the sterile and sterile stamens, and the sequence 619 bp, was cloned from the pollen fertile material. The fragment 395 bp. was cloned from the sterile stamen and the sterile unfertile stamen. Promoter analysis software NewPlace predicted that the promoter sequence contained TATA-box and CAAT-box, where TATA-box was located at -53 bp and -54 bp upstream of the initiation codon, and CAAT-box was located at -132 bp and -167 bp upstream of the start codon, respectively. The sequence also contains a large number of cis-acting elements, such as floral specific expression elements such as: GTGA,AGAAA and TGTGG and other homeotropic elements. 4. C4 gene was obtained by homologous cloning method. Combined with transcriptome data analysis, it was found that this gene had high homology with PHD- zinc finger protein white MMD1 gene, which controlled male sterility gene. A series of bioinformatics analysis showed that the highest homology of C4 gene and MMD1 was 57. The amino acid sequence of C4 gene had no transmembrane region and no signal peptide. The subcellular localization suggested that C4 gene was located in the nucleus. The domain contains PHD- zinc finger protein domain, and all the active sites are located in this region, which has the activity of zinc finger protease, which is consistent with the previous prediction, which lays the foundation for further study of this gene.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S682.29;Q943.2
，
本文編號：2280775
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