本文關(guān)鍵詞： 鹽生草屬 核型分析 耐鹽基因克隆 植物表達(dá)載體　出處：《甘肅農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：鹽脅迫是影響植物生長(zhǎng)發(fā)育,造成作物產(chǎn)量降低的主要非生物因素之一。它對(duì)植物的危害主要表現(xiàn)在滲透壓脅迫和離子脅迫等方面,使植物體細(xì)胞膜結(jié)構(gòu)、酶活性、光合作用、呼吸作用器官受到影響,且在植物體內(nèi)作用產(chǎn)生活性氧,導(dǎo)致氧化脅迫,使農(nóng)作物生長(zhǎng)失衡,產(chǎn)量降低甚至死亡。改良鹽堿地可通過改良土壤、合理灌溉等農(nóng)業(yè)措施,或利用基因工程技術(shù)培育耐鹽堿作物品種以提高作物的適應(yīng)性。鹽生草和白莖鹽生草都屬于藜科鹽生草屬一年生草本植物,分布于我國(guó)的甘肅西北部、新疆地區(qū)、青海地區(qū)及西藏地區(qū)等,是一種典型的鹽生植物。對(duì)這兩種植物的染色體核型進(jìn)行分析,為開展鹽生草基因組測(cè)序的準(zhǔn)確分析及我國(guó)鹽生植物的種類鑒別、親緣關(guān)系遠(yuǎn)近和種質(zhì)資源的遺傳多樣性提供細(xì)胞學(xué)依據(jù)。鹽生草植物地上有很多分枝的莖,具有保持水土、防止風(fēng)沙、耐鹽和抗旱能力,其自身蘊(yùn)含了很多的耐鹽基因,尤其在植物對(duì)鹽堿混合脅迫下基因表達(dá)差異性的表現(xiàn),極有可能挖掘大量與耐鹽堿方面的有利基因,對(duì)改良作物性狀及培育耐鹽堿作物具有重要意義。下列內(nèi)容為本實(shí)驗(yàn)的研究結(jié)果:1.鹽生草屬染色體核型分析通過對(duì)鹽生草屬的兩種鹽生植物進(jìn)行核型分析,其結(jié)果為:鹽生草和白莖鹽生草植物染色體數(shù)目均為18條,二倍體植物,染色體基數(shù)均為9,核型公式分別表示為2n=2x=18=12m+6sm和2n=2x=18=16m+2sm,都是由近中部著絲點(diǎn)區(qū)(sm)和中部著絲點(diǎn)區(qū)(m)構(gòu)成。相對(duì)長(zhǎng)度分別在7.808%-14.888%和7.50%-15.13%之間。核不對(duì)稱系數(shù)分別為61.908%和41.662%,其核型分別為“2A”和“1B”型,由結(jié)果可以看出白莖鹽生草屬于較原始的物種。2.相關(guān)耐鹽基因的克�、僖喳}生草cDNA為模板,利用RACE技術(shù)與RT-PCR方法獲得了Unigene19352和Unigene18968的3’端序列,經(jīng)過拼接得到3’端全長(zhǎng)序列分別為555 bp和608 bp,為后期cDNA全長(zhǎng)的獲得奠定了基礎(chǔ)。②采用RT-PCR方法從鹽生草中克隆到Unigene19095的cDNA全長(zhǎng)序列。分析結(jié)果為:此基因cDNA全長(zhǎng)為678 bp,包含3’UTR區(qū)388個(gè)核苷酸,5’UTR區(qū)29個(gè)核苷酸,開放閱讀框長(zhǎng)度261 bp,編碼86個(gè)氨基酸,分子量為9.67 kDa。軟件預(yù)測(cè)該基因編碼的蛋白存在信號(hào)肽,含有兩個(gè)跨膜區(qū)域,二級(jí)結(jié)構(gòu)中α螺旋,延伸鏈,隨機(jī)卷曲,β-轉(zhuǎn)角各占22.09%,36.05%,37.21%、4.65%,含有3個(gè)磷酸化位點(diǎn),但該基因的具體功能需要更深層次研究確定。3.植物表達(dá)載體CPB-Unigene19095的構(gòu)建及轉(zhuǎn)化成功構(gòu)建表達(dá)載體CPB-Unigene19095,并轉(zhuǎn)入農(nóng)桿菌LBA4404,采用蘸花法侵染擬南芥花序,獲得擬南芥抗性植株T1代。經(jīng)過PCR檢測(cè),初步確定Unigene19095已成功轉(zhuǎn)入擬南芥中,為進(jìn)一步得到純合的轉(zhuǎn)基因擬南芥植株以及其后代的表型檢測(cè),功能驗(yàn)證奠定了基礎(chǔ)。
[Abstract]:Salt stress is one of the main abiotic factors that affect the growth and development of plants and reduce crop yield. Its harm to plants is mainly reflected in osmotic stress and ion stress. Enzyme activity, photosynthesis and respiration are affected, and reactive oxygen species (Ros) are produced in plants, which lead to oxidative stress and make crops grow out of balance. The improvement of saline-alkali land can be done by improving soil, reasonable irrigation and other agricultural measures. In order to improve the adaptability of saline-tolerant crops, both halophyte and salt-stem halophytes belong to the annual herbaceous plants of the genus Chenopodiaceae, which are distributed in the northwest of Gansu Province. Xinjiang, Qinghai and Tibet are typical halophytes. For the accurate analysis of the genome sequencing of halophytes and the species identification of halophytes in China, the genetic diversity of germplasm resources and the genetic diversity of the genetic resources of halophytes were provided with cytological basis. There were many branched stems on the ground of halophytes. With the ability of soil and water conservation, sand prevention, salt tolerance and drought resistance, it contains a lot of salt tolerance genes, especially the difference of gene expression under saline-alkali mixed stress. It is highly likely to dig up a large number of favorable genes related to salt and alkali tolerance. It is of great significance to improve crop characters and cultivate saline-tolerant crops. The following contents are the results of this study: 1. Karyotype analysis of two halophytes of halophytes is carried out by means of karyotype analysis of halophytes. The results were as follows: the number of chromosomes of halophytes and albicans was 18, and the chromosome number of diploid plants was 9. The karyotype formula is expressed as 2ng ~ 2x ~ (18) 12m ~ 6sm and 2n ~ 2x ~ (18) ~ (18) ~ (16) m ~ (2sm) respectively. Both from the near central metacentric area (Sm) and from the central centromere region (Mm). The relative length was between 7.808% -14.88% and 7.50-15.13%. The nuclear asymmetry coefficient was 61.908% and 41.662%, respectively. The karyotypes were "2A" and "1B", respectively. From the results, it can be seen that the halophytes belong to the more primitive species. 2. Clone 1 of the related salt tolerance gene takes cDNA as the template. The 3'end sequences of Unigene19352 and Unigene18968 were obtained by using RACE technique and RT-PCR method. The full-length sequence of 3 'end was 555 BP and 608 BP, respectively. 2. The whole cDNA sequence of Unigene19095 was cloned from halophytic grass by RT-PCR method. The results were as follows:. The cDNA length of the gene is 678bp. There are 388 nucleotides in UTR region and 29 nucleotides in 5 UTR region. The open reading frame is 261 BP long and encodes 86 amino acids. The molecular weight of the protein was 9.67 kDa.The software predicted that the protein encoded by the gene contained two transmembrane regions, a helix in the secondary structure, extended chains and random curls. The 尾 -turning angle accounted for 22.09% and 36.05%, and 37.21%, 4.65%, containing three phosphorylated sites. However, the specific function of the gene needs to be further studied and determined. 3. The construction and transformation of plant expression vector CPB-Unigene19095 and the successful construction of expression vector CPB-Unigene1. 9095. And transferred to Agrobacterium tumefaciens LBA4404, infected Arabidopsis thaliana inflorescence with the method of dipping flowers, and obtained T1 generation of Arabidopsis resistant plants. The results were detected by PCR. It was preliminarily confirmed that Unigene19095 had been successfully transferred into Arabidopsis thaliana, which laid a foundation for the phenotypic detection and functional verification of homozygous transgenic Arabidopsis thaliana plants and their progenies.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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