【摘要】：該試驗(yàn)采用RT-PCR和RACE技術(shù),對(duì)龍眼多糖合成的關(guān)鍵基因尿苷二磷酸-葡萄糖6-脫氫酶基因(DlUGD6)進(jìn)行分離克隆、生物信息學(xué)分析和亞細(xì)胞定位研究,并采用qRT-PCR技術(shù),對(duì)其在龍眼體細(xì)胞胚胎發(fā)生、合子胚發(fā)育及不同組織器官中的表達(dá)模式進(jìn)行分析。結(jié)果表明:(1)DlUGD6基因的cDNA序列全長(zhǎng)1 860bp,包含開(kāi)放閱讀框1 443bp,編碼480個(gè)氨基酸(GenBank登錄號(hào)KU198438);生物信息學(xué)分析顯示,DlUGD6屬于穩(wěn)定的酸性親水蛋白,不含信號(hào)肽,具有跨膜結(jié)構(gòu)和3個(gè)典型的保守結(jié)構(gòu)域,屬于UDP-葡萄糖/GDP-甘露糖脫氫酶家族;進(jìn)化樹(shù)分析表明,DlUGD6與柑橘親緣關(guān)系較近。(2)洋蔥內(nèi)表皮GFP熒光定位觀察發(fā)現(xiàn),DlUGD6定位于細(xì)胞質(zhì);qRT-PCR結(jié)果顯示,DlUGD6在龍眼非胚性愈傷組織中表達(dá)量相對(duì)較高,且在其他體胚發(fā)育階段也均有穩(wěn)定表達(dá);在合子胚發(fā)育中子葉胚形成后第8天(S3)和第24天(S7)時(shí)表達(dá)量最高,整體呈"W"型;在不同組織器官中,DlUGD6在花藥和莖中的表達(dá)量最高,且整體上生殖器官中的表達(dá)水平高于營(yíng)養(yǎng)器官。研究認(rèn)為,DlUGD6基因可能參與龍眼生長(zhǎng)發(fā)育各個(gè)階段中細(xì)胞壁多糖合成。
[Abstract]:The key gene of longan polysaccharide synthesis was isolated and cloned by RT-PCR and RACE, bioinformatics analysis and subcellular localization were carried out, and qRT-PCR technique was used. Its expression patterns in somatic embryogenesis, zygotic embryo development and different tissues and organs of longan were analyzed. The results showed that: (1) the cDNA sequence of DlUGD6 gene was 1 860 BP, including open reading frame 1443 BP, encoding 480 amino acids (GenBank accession number KU198438); Bioinformatics analysis showed that DlUGD6 was a stable acidic hydrophilic protein with no signal peptide, and had transmembrane structure and three typical conserved domains, belonging to the UDP- glucose / GDP- mannose dehydrogenase family. Phylogenetic tree analysis showed that DlUGD6 was closely related to citrus. (2) GFP fluorescence localization of onion inner epidermis showed that DlUGD6 was located in cytoplasm; QRT-PCR results showed that the expression of DlUGD6 was relatively high in non-embryogenic callus of longan and stable in other somatic embryogenesis. At the 8th (S3) and 24th (S7) days after the development of zygotic embryos, the highest expression level was observed, and the whole expression was "W" type. The expression of DlUGD6 in anthers and stems was the highest in different tissues and organs, and the overall expression level of DlUGD6 in reproductive organs was higher than that in vegetative organs. It is suggested that DlUGD6 gene may be involved in the synthesis of polysaccharides from the cell wall of longan in various stages of growth and development.
【作者單位】： 福建農(nóng)林大學(xué)園藝植物生物工程研究所;
【基金】：國(guó)家自然科學(xué)基金(31572088,31272149) 福建省重大科技專項(xiàng)(2015NZ-0002-1)
【分類號(hào)】：Q943.2;S667.2
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本文編號(hào)：2323924