發(fā)布時(shí)間：2017-12-28 03:28

  本文關(guān)鍵詞：兩種暖季型草坪草對(duì)低溫的響應(yīng)機(jī)制及ZjADH基因的克隆與功能分析　出處：《北京林業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 日本結(jié)縷草 普通狗牙根 低溫生理應(yīng)答 ZjADH基因 功能分析

【摘要】：日本結(jié)縷草及普通狗牙根均為典型的暖季型草坪草,在我國(guó)南北地區(qū)均有分布。低溫傷害是暖季型草坪草在北方地區(qū)應(yīng)用時(shí)經(jīng)常遇到的一種災(zāi)害,它不僅會(huì)導(dǎo)致草坪草的生長(zhǎng)受到損害,甚至?xí)鹬仓晁劳觥Ｑ芯康蜏孛{迫對(duì)暖季型草坪草的傷害機(jī)理,探索其抗寒機(jī)制具有重要的理論和實(shí)際應(yīng)用意義。ADH基因在植物抵抗逆境脅迫包括低溫逆境起著重要的作用,分離并分析日本結(jié)縷草ADH基因?qū)τ诟玫睦斫饨Y(jié)縷草低溫響應(yīng)起著重要的作用。本研究以普通狗牙根和日本結(jié)縷草的匍匐莖為材料,研究二者在低溫脅迫條件下相關(guān)生理指標(biāo)及分子指標(biāo)的動(dòng)態(tài)變化,探討寒冷脅迫對(duì)其生理及分子層面的影響；針對(duì)抗寒性較強(qiáng)的日本結(jié)縷草,設(shè)計(jì)引物克隆日本結(jié)縷草ADH基因全長(zhǎng)序列,將其命名為ZjADH,對(duì)其進(jìn)行序列分析和功能分析。研究結(jié)果如下：1.低溫脅迫下,草坪表觀質(zhì)量下降,兩種草坪草的葉綠素含量、類胡蘿卜素含量、光化學(xué)效率逐漸下降,脯氨酸、可溶性糖及可溶性蛋白逐漸積累,電解質(zhì)滲透率及丙二醛含量逐漸上升,且低溫鍛煉能顯著降低日本結(jié)縷草和普通狗牙根的低溫半致死溫度。傷害指標(biāo)評(píng)價(jià)結(jié)果表明日本結(jié)縷草抗寒性強(qiáng)于普通狗牙根。2.隨著低溫脅迫時(shí)間的增長(zhǎng),日本結(jié)縷草和普通狗牙根的過(guò)氧化氫及超氧陰離子含量逐漸增加,SOD和POD活性逐漸升高,APX活性則先升高后降低。CytSOD、 MnSOD、POD、APX基因表達(dá)量出現(xiàn)上調(diào),CAT及ChlSOD基因表達(dá)量則逐漸下降,FeSOD表達(dá)量則先下降再升高。研究表明,低溫脅迫下,植株啟動(dòng)抗氧化機(jī)制,清除活性氧,降低植物損傷。3.低溫脅迫下,兩種草坪草的ABA含量呈先升高再降低,CTK含量則逐漸下降。隨著脅迫時(shí)間的增長(zhǎng),LEA基因表達(dá)量逐漸上調(diào),RAB基因表達(dá)量先升高再下降。內(nèi)源激素的變化及低溫誘導(dǎo)蛋白的表達(dá)對(duì)于激活響應(yīng)機(jī)制,提高植株抗寒性具有重要作用。4.設(shè)計(jì)用于RACE反應(yīng)的兩條引物,進(jìn)行3'RACE和5'RACE擴(kuò)增,得到765bp和816bp的片段。通過(guò)序列拼接獲得該基因的cDNA,序列分析該基因開(kāi)放閱讀框?yàn)?140-bp,編碼一條含有379個(gè)氨基酸的多肽鏈。將該氨基酸序列進(jìn)行Blast比較,構(gòu)建ADH蛋白的系統(tǒng)發(fā)育樹(shù),發(fā)現(xiàn)日本結(jié)縷草與溝葉結(jié)縷草的ADH親緣關(guān)系最近。克隆的基因?yàn)槿毡窘Y(jié)縷草ADH基因,命名為ZjADH, Genbank中登陸號(hào)為KT596065。5.通過(guò)實(shí)時(shí)定量RT-PCR方法分析了ZjADH基因在日本結(jié)縷草不同組織中轉(zhuǎn)錄水平上的表達(dá)情況。結(jié)果顯示,ZjADH基因在根中表達(dá)量最高,在葉中表達(dá)量最低；ZjADH在低溫、干旱和高鹽脅迫條件下表達(dá)量升高,并且可受到ABA、MeJA和SA信號(hào)誘導(dǎo)。6.將含有完整開(kāi)放性閱讀框的ZjADH基因片段插入3302-YFP載體,構(gòu)建了融合蛋白表達(dá)載體ZjADH-YFP,注射法轉(zhuǎn)化煙草葉肉細(xì)胞后,利用激光共聚焦顯微鏡觀察發(fā)現(xiàn)該蛋白定位于細(xì)胞質(zhì)。7.構(gòu)建植物表達(dá)載體3302-ZjADH,轉(zhuǎn)入到農(nóng)桿菌GV3101中,通過(guò)花序浸泡法,轉(zhuǎn)化野生型擬南芥植株。經(jīng)草銨膦篩選得到具有草銨膦抗性的幼苗,提取抗性擬南芥葉片DNA,進(jìn)行PCR檢測(cè),證實(shí)成功獲得轉(zhuǎn)基因植株。提取轉(zhuǎn)基因植株中的總RNA,通過(guò)RT-PCR檢測(cè),結(jié)果顯示,轉(zhuǎn)基因擬南芥能夠高效表達(dá)ZjADH基因。8.對(duì)轉(zhuǎn)基因擬南芥進(jìn)行抗性檢測(cè),結(jié)果顯示：轉(zhuǎn)基因擬南芥植株對(duì)低溫的抗性明顯高于野生型擬南芥；轉(zhuǎn)基因擬南芥中與脅迫相關(guān)的基因表達(dá)水平明顯高于野生型擬南芥植株。對(duì)轉(zhuǎn)基因植株進(jìn)行生理生化分析,結(jié)果顯示：轉(zhuǎn)基因植物的脯氨酸含量高于野生型擬南芥。本研究表明,低溫脅迫下,日本結(jié)縷草和普通狗牙根通過(guò)滲透調(diào)節(jié)物質(zhì)積累、抗氧化代謝、內(nèi)源激素及低溫誘導(dǎo)蛋白的表達(dá),提高植株抗寒性,且日本結(jié)縷草抗寒性強(qiáng)于普通狗牙根。以抗寒性較強(qiáng)的日本結(jié)縷草為材料,獲得了日本結(jié)縷草ADH基因,全長(zhǎng)1140bp,定位于細(xì)胞質(zhì),該基因在根中表達(dá)量最高,在逆境脅迫條件下,表達(dá)量升高顯著,成功獲得了轉(zhuǎn)基因擬南芥植株,轉(zhuǎn)基因擬南芥對(duì)低溫抗性與野生型比起來(lái)明顯提高,初步證明日本結(jié)縷草ADH基因與植物逆境響應(yīng)及抗寒性有關(guān)。
[Abstract]:Zoysia japonica and common dog root are typical warm season turfgrass, which are distributed in the north and south of China. Low temperature injury is a common calamity often encountered in the application of warm season turfgrass in the northern area. It will not only cause damage to the growth of turfgrass, but even cause plant death. It is of great theoretical and practical significance to study the damage mechanism of low temperature stress on warm season turfgrass and explore its cold resistance mechanism. ADH gene plays an important role in plant resistance to stress, including low temperature stress. Isolation and analysis of ADH gene from Zoysia japonica plays an important role in better understanding of low temperature response of Zoysia japonica. In this study, Common Bermudagrass and zoysiagrass stolon as materials, study of the two dynamic changes in physiological indexes and molecular indexes under low temperature stress, cold stress to investigate the effects on the physiological and molecular level; the cold resistance of Zoysia japonica strong, primers were designed to clone the full-length sequence of Japan. Zoysia ADH gene, named ZjADH, analyze sequence and function of the. The results are as follows: 1. under low temperature stress, the apparent decline in the quality of the lawn, the chlorophyll content, two kinds of lawn grass carotenoid content, photochemical efficiency decreased, proline, soluble sugar and soluble protein accumulation, membrane permeability and MDA content increased gradually, and the low temperature exercise can significantly reduce the Z.japonica and ordinary Bermuda semilethal temperature. The result of injury index evaluation showed that the cold resistance of Zoysia japonica was stronger than that of common dog root. 2., with the increase of low temperature stress time, the content of hydrogen peroxide and superoxide anion increased gradually, and the activity of SOD and POD increased gradually, while the activity of APX increased first and then decreased. The expression of CytSOD, MnSOD, POD and APX was up-regulated, while the expression of CAT and ChlSOD decreased gradually, while the expression of FeSOD decreased first and then increased. The study showed that under low temperature stress, the plant started antioxidant mechanism, scavenging reactive oxygen species and reducing plant damage. 3. under low temperature stress, the ABA content of two turf grass increased first and then decreased, while the content of CTK decreased gradually. With the increase of stress time, the expression of LEA gene increased gradually, and the expression of RAB gene increased first and then decreased. The changes of endogenous hormones and the expression of low temperature induced proteins play an important role in activating the response mechanism and improving the cold resistance of plants. 4. the two primers used for RACE reaction were designed to amplify 3'RACE and 5'RACE, and the fragments of 765bp and 816bp were obtained. The cDNA of the gene was obtained by sequence splicing, and the sequence analysis of the gene opened the open reading frame to 1140-bp and encodes a polypeptide chain containing 379 amino acids. The phylogenetic tree of the ADH protein was constructed by comparing the amino acid sequence with Blast, and the closest relationship between Zoysia japonica and the ADH of Zoysia japonica was found. The cloned gene is the ADH gene of Zoysia japonica, named ZjADH, and the landfall number in Genbank is KT596065. 5. the expression of ZjADH gene in the transcriptional level of Zoysia japonica was analyzed by real-time quantitative RT-PCR method. The results showed that the expression level of ZjADH gene in roots was the highest, and the expression level in leaves was the lowest. ZjADH expression was increased under low temperature, drought and high salt stress, and it could be induced by ABA, MeJA and SA signals. 6., insert the ZjADH gene fragment containing a complete open reading frame into the 3302-YFP vector, construct the fusion protein expression vector ZjADH-YFP, and transform it into tobacco mesophyll cells by injection, then we observed the localization of the protein in cytoplasm by laser scanning confocal microscopy. 7. the plant expression vector 3302-ZjADH was constructed and transferred into Agrobacterium tumefaciens GV3101 to convert wild type Arabidopsis plants through inflorescence soaking. With glufosinate resistance seedlings by glufosinate screening, extraction resistance of Arabidopsis thaliana leaves DNA, PCR detection, transgenic plants were obtained successfully. The total RNA in transgenic plants was extracted and detected by RT-PCR. The results showed that transgenic Arabidopsis could express the ZjADH gene efficiently. 8., resistance detection of transgenic Arabidopsis thaliana showed that the resistance of transgenic Arabidopsis plants to low temperature was significantly higher than that of wild Arabidopsis thaliana, and the expression level of stress related genes in transgenic Arabidopsis thaliana was significantly higher than that in wild Arabidopsis thaliana plants. The results of physiological and biochemical analysis of transgenic plants showed that the proline content of transgenic plants was higher than that of wild Arabidopsis. This study showed that under low temperature stress, Japanese Zoysia and Common Bermudagrass increased plant cold tolerance through osmotic adjustment substance accumulation, antioxidant metabolism, endogenous hormones and low temperature induced protein expression, and the cold resistance of Zoysia japonica was stronger than that of Common Bermudagrass. The cold resistance of Zoysia japonica as strong material, obtained the Zoysia japonica ADH gene, full-length 1140bp, localized in the cytoplasm, the gene expression level in root was highest in the stress condition, the expression level increased significantly, successfully obtained the transgenic Arabidopsis plants, transgenic Arabidopsis wild and resistance to low temperature type than increased significantly, preliminary evidence of Zoysia japonica ADH gene related to plant stress response and cold resistance.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S688.4

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 樊曉莉,包滿珠;組織培養(yǎng)獲得日本結(jié)縷草綠色期延長(zhǎng)株系[J];園藝學(xué)報(bào);2005年03期

2 齊春輝,梁小紅,韓烈保;日本結(jié)縷草胚性愈傷誘導(dǎo)與植株再生(簡(jiǎn)報(bào))[J];草地學(xué)報(bào);2005年03期

3 張俊衛(wèi),唐蜻,包滿珠;日本結(jié)縷草植株再生體系的研究[J];草業(yè)學(xué)報(bào);2005年02期

4 齊春輝,韓烈保,黃麟,方文娟;幾種因素對(duì)日本結(jié)縷草愈傷組織誘導(dǎo)與生長(zhǎng)影響的研究[J];四川草原;2005年04期

5 杜敏華;柴春月;;日本結(jié)縷草離體培養(yǎng)及高頻率植株的再生[J];東北林業(yè)大學(xué)學(xué)報(bào);2007年10期

6 孫建明;李惠英;婁燕宏;黃炳茹;;不同緯度種群日本結(jié)縷草對(duì)冷脅迫的生理反應(yīng)[J];草原與草坪;2010年06期

7 劉擁海;俞樂(lè);崔鐵成;;日本結(jié)縷草愈傷組織的誘導(dǎo)和植株再生[J];草業(yè)科學(xué);2010年03期

8 張巨明;趙鳴;;蘭引3號(hào)草坪型結(jié)縷草與日本結(jié)縷草的比較研究[J];草業(yè)科學(xué);1996年02期

9 張俊衛(wèi);唐蜻;包滿珠;;日本結(jié)縷草成熟種子愈傷組織誘導(dǎo)及植株再生的影響因子分析[J];中國(guó)農(nóng)業(yè)科學(xué);2006年02期

10 劉莉;包滿珠;;日本結(jié)縷草體細(xì)胞變異表型和分子標(biāo)記的研究[J];草業(yè)科學(xué);2012年12期

相關(guān)會(huì)議論文 前2條

1 程曉霞;代小梅;韓烈保;;日本結(jié)縷草隱花色素基因(Cry1a)的克隆及RNAi載體構(gòu)建[A];全國(guó)植物分子育種研討會(huì)摘要集[C];2009年

2 代小梅;;日本結(jié)縷草總RNA提取和mRNA分離方法的優(yōu)化[A];全國(guó)植物分子育種研討會(huì)摘要集[C];2009年

相關(guān)重要報(bào)紙文章 前2條

1 馬其東;北方寒冷氣候區(qū)（上）[N];中國(guó)花卉報(bào);2004年

2 記者 方林;“綠亞”承建加納足球場(chǎng)獲好評(píng)[N];中國(guó)花卉報(bào);2007年

相關(guān)博士學(xué)位論文 前7條

1 梁小紅;兩種暖季型草坪草對(duì)低溫的響應(yīng)機(jī)制及ZjADH基因的克隆與功能分析[D];北京林業(yè)大學(xué);2016年

2 方文娟;日本結(jié)縷草（Zoysia japonica Steud.）懸浮再生體系的建立與超低溫保存研究[D];北京林業(yè)大學(xué);2008年

3 程曉霞;GA_3調(diào)控日本結(jié)縷草葉片衰老過(guò)程相關(guān)基因的分離與初步功能分析[D];北京林業(yè)大學(xué);2010年

4 劉莉;日本結(jié)縷草IPT基因遺傳轉(zhuǎn)化及野牛草實(shí)生群體內(nèi)遺傳多樣性研究[D];華中農(nóng)業(yè)大學(xué);2010年

5 齊春輝;日本結(jié)樓草（Zoysia japonica Steud.）植株再生體系建立與DREB1A基因轉(zhuǎn)化研究[D];北京林業(yè)大學(xué);2005年

6 張磊;幾種草坪草轉(zhuǎn)基因及體細(xì)胞無(wú)性系變異的研究[D];安徽農(nóng)業(yè)大學(xué);2005年

7 代小梅;日本結(jié)縷草（Zoysia japonica Steud.）ZjGA20ox基因表達(dá)模式及RNA干擾載體遺傳轉(zhuǎn)化體系研究[D];北京林業(yè)大學(xué);2013年

相關(guān)碩士學(xué)位論文 前10條

1 可祥;日本結(jié)縷草ZjDREB2.2基因克隆及功能研究[D];中央民族大學(xué);2015年

2 張迷離;日本結(jié)縷草對(duì)低溫及外源激素的生理響應(yīng)研究[D];北京林業(yè)大學(xué);2013年

3 樊曉莉;日本結(jié)縷草植株再生體系優(yōu)化及根癌農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化的初步研究[D];華中農(nóng)業(yè)大學(xué);2005年

4 殷萍萍;日本結(jié)縷草響應(yīng)立枯絲核菌侵染及其轉(zhuǎn)錄組學(xué)研究[D];北京林業(yè)大學(xué);2015年

5 李亞超;日本結(jié)縷草在機(jī)械損傷脅迫下的轉(zhuǎn)錄組測(cè)序及分析[D];北京林業(yè)大學(xué);2015年

6 唐蜻;日本結(jié)縷草成熟胚愈傷誘導(dǎo)及植株再生的研究[D];華中農(nóng)業(yè)大學(xué);2003年

7 杜艷華;日本結(jié)縷草Na~+/H~+逆向轉(zhuǎn)運(yùn)蛋白基因的克隆及功能研究[D];華東師范大學(xué);2009年

8 閆美玲;日本結(jié)縷草再生體系優(yōu)化和根癌農(nóng)桿菌介導(dǎo)PSAG12-IPT基因的轉(zhuǎn)化研究[D];華中農(nóng)業(yè)大學(xué);2007年

9 劉穎;日本結(jié)縷草對(duì)低溫脅迫的生理響應(yīng)及抗寒相關(guān)性狀的QTL分析[D];南京農(nóng)業(yè)大學(xué);2010年

10 李云霞;H_2O_2誘導(dǎo)日本結(jié)縷草細(xì)胞程序性死亡及GA3緩解作用研究[D];北京林業(yè)大學(xué);2009年

，

本文編號(hào)：1344406