【作者】 张妙霞；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 果树学， 2010， 博士

【摘要】 本研究以福建三明野生香蕉(Musa spp., AB group, from Sanming City)叶片为材料,分别进行常温和低温胁迫下的几丁质酶基因和β-1,3-葡聚糖酶基因克隆,并采用实时荧光定量PCR技术分析常温和低温胁迫过程中其表达规律,以期获得抗冻蛋白基因,进一步了解其在常温和低温胁迫过程中的表达差异。主要研究结果如下：1三明野生香蕉低温胁迫下几丁质酶基因(ChiⅠ1、ChiⅠ2) cDNA全长的克隆及生物信息学分析以三明野生香蕉叶片为材料,采用RT-PCR结合RACE方法,获得了低温胁迫处理(4℃处理64h)下三明野生香蕉叶片几丁质酶基因的cDNA全长为1097bp,3’UTR为149bp, 3’poly(A)尾长17bp,将此基因命名为Chi 12(登录号：FJ222750)；同时还获得了常温(28℃)下三明野生香蕉叶片几丁质酶基因的cDNA全长为1115bp,5’UTR为14bp,3’UTR为177bp,3’poly(A)尾长17bp,将此基因命名为ChiⅠ1(登录号：FJ858155)。这两个序列与登录GenBank的其它植物几丁质酶基因有很高的同源性。应用生物信息学方法分析结果表明：ChiⅠ1蛋白和ChiⅠ2蛋白均为酸性的亲水性跨膜蛋白,亚细胞定位为胞外空间,属于糖苷水解酶19家族成员,为Ⅰ类几丁质酶。ChiⅠ2蛋白具有两个几丁质结合区,这可能是其兼有抗冻活性及抗病菌活性的主要功能区域。三明野生香蕉ChiⅠ2基因与国际上已经报道的植物几丁质酶抗冻蛋白基因的结构很相似,初步推断ChiⅠ2基因是几丁质酶抗冻蛋白基因。2三明野生香蕉ChiⅠ1与ChiⅠ2基因DNA序列的克隆从DNA水平克隆得到三明野生香蕉ChiⅠ1与ChiⅠ2基因,均有ATG起始密码子和TAG终止密码子。ChiⅠ2基因DNA序列长1183bp(登录号：GU391234)；ChiⅠ1基因DNA序列长1159bp(登录号：GU391235)。内含子分析表明ChiⅠ1与ChiⅠ2基因均由4个外显子和3个内含子组成,所有内含子的剪切位点均符合真核生物GT-AG规则。ChiⅠ2基因与ChiⅠ1基因相比第一个外显子增加了24bp,其余外显子与内含子部分均没有差别。这增多的24bp的外显子可能是几丁质酶抗冻蛋白基因的作用位点。3三明野生香蕉ChiⅠ1与ChiⅠ2基因启动子的克隆采用交错式热不对称PCR染色体步移方法克隆三明野生香蕉ChiⅠ1和ChiⅠ2基因部分启动子序列。ChiⅠ1基因启动子长323bp(登录号：GU391235); ChiⅠ2基因启动子长846bp(登录号：GU391234)。ChiⅠ1基因可能存在的基础启动子区域为第272-322处,其序列为：5’-GGCAGGAATCCATTTCTCTATATAAGCACCACCTCCCACCCACACCACCA-3’。预测转录起始点在第312bp处的C,含有与光反应、水杨酸响应、高转录水平及分生组织表达相关的顺式作用调控元件。Chi 12基因在第522-572bp处存在可能的基础启动子区域,其序列为：5’-GCAATTTATTTTAAAAAAAACTCTTGTGATAAGCTTTTATACCCTCATAT-3’。预测转录起始点在第562bp处的A,含有与光反应、厌氧感应、真菌诱发子、MeJA响应、赤霉素响应、水杨酸响应、高转录水平及生理节奏控制等相关的顺式作用调控元件。其中真菌诱发子响应要素对真菌诱导极其重要,主要存在于病程相关基因的启动子中,也是所在启动子的应激反应元件；而MeJA响应元件与抗寒伤害有关,说明三明野生香蕉Chi 12基因可能是既具有抗病又具有抗寒特性的几丁质酶抗冻蛋白基因。4三明野生香蕉低温胁迫下几丁质酶基因的表达分析采用18S rRNA基因作为内参基因,利用实时荧光定量PCR技术分析三明野生香蕉低温胁迫过程中ChiⅠ1基因与ChiⅠ2基因的转录水平表达变化。分析结果显示：ChiⅠ2基因在15℃处理30h时表达量达到最高,当温度达到4℃时,ChiⅠ2基因的表达量随胁迫时间的延长呈现先升后降再升的趋势；ChiⅠ1基因的相对表达量在4℃处理24h时达到最大值,整体表达趋势呈“M”型,很明显可以看出ChiⅠ2基因对温度变化敏感。一般认为15℃是影响香蕉生长的临界温度,当三明野生香蕉在15℃处理条件下,ChiⅠ2基因则迅速感应到低温伤害,大量表达；4℃低温表达时表明ChiⅠ2基因具有累积效应。而ChiⅠ1基因的表达表明其对温度和低温时间不敏感,只有当低温持续时间达到一定程度时,才诱发其表达。以上可以进一步说明三明野生香蕉ChiⅠ2基因是几丁质酶抗冻蛋白基因,而Chi I 1是Chi 12的同工酶。对三明野生香蕉外切和内切几丁质酶活性的研究发现两者均在4℃处理64h时达到酶活性的最高点,这可能是具有抗冻活性的几丁质酶基因在低温处理过程中表达的结果。5三明野生香蕉低温胁迫下β-1,3-葡聚糖酶基因(gsp、gsp1) cDNA全长的获得及生物信息学分析以三明野生香蕉叶片为材料,采用RT-PCR结合RACE方法,获得了低温胁迫处理(4℃处理64h)下三明野生香蕉叶片β-1,3-葡聚糖酶基因cDNA的ORF序列,全长915bp,编码304个氨基酸,命名为：gsp(登录号：FJ858156)；同时还获得了常温(28℃)下三明野生香蕉叶片β-1,3-葡聚糖酶基因cDNA的ORF序列,全长951bp,编码316个氨基酸,命名为：gsp1(登录号：HQ018802)。gsp基因与gsp1基因序列与登录GenBank的其它香蕉品种的β-1,3-葡聚糖酶基因有很高的同源性。应用生物信息学方法分析结果表明：GSP蛋白和GSP1蛋白属于糖基水解酶第十七家族,是一种碱性β-1,3-葡聚糖酶,分别定位于细胞质与质膜上；GSP蛋白属于亲水蛋白,GSP1蛋白属于疏水蛋白,且gsp基因与gspl基因相比少了36bp,主要在N端,初步推测gsp基因可能是抗冻蛋白基因。6三明野生香蕉gsp与gsp1基因DNA序列的克隆从DNA水平克隆得到三明野生香蕉gsp与gspl基因,gsp基因基因组DNA序列长1540bp(登录号:HQ018804)。gsp1基因的基因组DNA序列长1531bp(登录号为：HQ018803)。内含子分析显示gsp基因内含子剪切不符合传统的GT-AG法则,含有一个625bp的内含子；而gsp1基因内含子剪切符合GT-AG法则,含有一个580bp的内含子。内含子的差异可能是β-1,3-葡聚糖酶抗冻蛋白基因与一般β-1,3-葡聚糖酶基因间的差异引起的。7三明野生香蕉低温胁迫下β-1,3-葡聚糖酶基因的表达采用18S rRNA基因作为内参基因,利用实时荧光定量PCR技术分析三明野生香蕉低温处理过程中gsp与gspl基因的转录水平表达变化。分析结果显示：三明野生香蕉叶片gsp基因在15℃处理30h时的相对表达量达到最大值,整体表达趋势与ChiⅠ2基因相似,而gspl基因在整个低温胁迫处理过程中实时表达量的变化不大,这一现象说明三明野生香蕉gsp基因与ChiⅠ2基因均能对低温胁迫做出快速应激反应,且具有累积效应,进一步说明gsp基因可能是β-1,3-葡聚糖酶抗冻蛋白基因。总之,本研究从具有抗寒特性的三明野生香蕉叶片中克隆到低温胁迫的几丁质酶基因和β-1,3-葡聚糖酶基因,根据基因的分子量、结构及其在低温胁迫下的表达等推断三明野生香蕉ChiⅠ2基因与gsp基因很可能是抗冻蛋白基因,为解决香蕉及其他热带亚热带作物抗寒育种提供目的基因；同时还克隆到了ChiⅠ1与ChiⅠ2基因启动子部分序列,有利于进一步了解野生香蕉的抗寒机制及抗寒基因的调控表达机制。更多还原

【Abstract】 In the experiment, the leaves of the wild banana (Musa spp., AB group, from Sanming City) under normal temperature and cold stress were used as materials for cloning the chitinase andβ-1,3-glucanase genes for obtaining antifreeze protein genes. The rules of gene expression during normal temperature and cold stress were analyzed by real time fluorescence quantitative PCR (q-PCR) for comfirming antifreeze protein genes and revealing the expression difference of the two genes during normal temperature and cold stress. The main results were as follows:1 Cloning and bioinformatics analysis of the cDNA of chitinase genes(ChiⅠ1 and ChiⅠ2) of the wild banana from Sanming City under cold stressFull length cDNA (1097bp) of the chitinase gene was cloned from the wild banana leaves under cold stress (4℃64h) by RT-PCR combined with RACE. It contained 149bp in the 3’UTR and 3’-end including 17bp poly (A) tail, named as ChiⅠ2 (GenBank:FJ222750). Meanwhile, a full length of cDNA (1115bp) of chitinase gene was cloned from the wild banana leaves under the normal temperature (28℃), containing 14bp 5’UTR and 149bp 3’UTR and 3’-end including 17bp poly (A) tails, named as ChiⅠ1 (GenBank:FJ858155). The above two genes were highly homologous to corresponding genes of other plants reported in the database of NCBI. The results which were analyzed by using bioinformatic methods showed that the two proteins were acidic hydrophilic proteins with transmembrane domain and mainly located outside. They belonged to the chitinases family 19, chitinase classⅠ. ChiⅠ2 contained two chitin-binding domains, which maybe the main function domains combining antifreeze activity with antivirus activity. The structure of Chi 12 gene of the wild banana from Sanming City was similar to chitinase antifreeze protein which had reported in the world. Accordingly, Chi 12 gene was preliminary inferred to as a chitinase-AFP gene.2 The cloning of ChiⅠ1 and ChiⅠ2 genomics of the wild banana from Sanming CityThe DNA sequences of ChiⅠ1 and ChiⅠ2 genes of the wild banana from Sanming City were cloned, which ATG was the initiation codon and TAG was the stop codon. The full sequences of Chi 12 were 1183bp (GenBank:GU391234) and the full sequences of ChiⅠ1 were 1159bp (GenBank:GU391235). Sequence analyses indicated that ChiⅠ1 and ChiⅠ2 genes both contained four exons and three introns, which splicing sites obeyed with the GT-AG rule. The first exon of Chi 12 has 24bp more than the first exon of ChiⅠ1, which maybe the antifreeze site of chitinase-AFP. There was no difference of other exons and introns between ChiⅠ1 and Chi 12.3 Promoter cloning of ChiⅠ1 and ChiⅠ2 of the wild banana from Sanming CityThe promoter sequences of ChiⅠ1 and ChiⅠ2 of the wild banana from Sanming City were cloned by genomic walking methods of thermal asymmetric inter-laced PCR. The length of ChiⅠ1 promoter was 323bp (GenBank:GU391235) and the length of Chi 12 promoter was 846bp (GenBank:GU391234). The promoter of ChiⅠ1 had a likehood of basic promoter area from 272bp to 322bp with a predicted starting site of transcription as Cytonsine on the 312th base, and it contained some cis-action elements such as a module involved in light responsiveness, cis-acting element involved in salicylic acid responsiveness, and a cis-acting element conferring high transcription levels. The promoter of Chi 12 also had a possible basic promoter area from 522bp to 572bp with a predicted starting site, adeine on the 562th, containing some cis-action elements such as a module involved in light responsiveness, cis-acting regulatory element essential for anaerobic induction, fungal elicitor responsive element, cis-acting regulatory element involved in the MeJA-responsiveness, gibberellin-responsive element, cis-acting element involved in salicylic acid responsiveness, cis-acting element conferring high transcription levels and cis-acting regulatory element involved in circadian control. The fungal elicitor responsive element, which mainly existed in the promoter of pathogenesis-related protein genes and the element of promoter stress response, was very critical for inducing fungal response. The element was involved in MeJA-responsiveness associated with cold hardiness, which indicated that Chi 12 of the wild banana from Sanming City maybe a chitinase antifreeze protein gene relative to cold resistance and antivirus.4 The expression of chitinase gene of the wild banana from Sanming City under low temperature stressUsing 18S rRNA as the reference gene, the expressions of ChiⅠ1 and ChiⅠ2 under cold stress were analyzed by q-PCR. The results showed that Chi 12 had the most abundant expression under treatment of 15℃for 30h. At 4℃, the expression of Chi 12 increased at the beginning of the cold stress treatment, then declined and finally increased at the end of 24h cold stress treatment. ChiⅠ1 had the most abundant expression under 24h 4℃treatment, with the whole procession of ChiⅠ1 expression presented as a ’M’ curve. The results confirmed that Chi 12 was sensitive to temperature variation. Generally, the critical temperature of banana growing was 15℃. When the wild banana was treated under 15℃, Chi 12 gene responded to injury via low temperature, as seen in the high expression levels. The expression under 4℃indicated the Chi 12 gene had a cumulative effect. The expression showed that ChiⅠ1 gene was insensitive to temperature and cold stress time, only when the low temperature continued for some time, was the gene expressed. All these showed that Chi 12 gene of the wild banana was a chitinase-AFP gene, and ChiⅠ1was the isozyme of ChiⅠ2.Also, activity of exo-chitinase and endo-chitinase from the wild banana were measured, the results showed the activity of both genes were strongest after 64h 4℃treatment, which was supposed to be induced by the expression of the antifreeze gene from the chitinase family under cold stress.5 cDNA cloning and bioinformatics analysis ofβ-1,3-glucanase genes (gsp and gspl) of the wild banana from Sanming City under cold stressFrom the wild banana leaves under 64h 4℃treatment, the ORF ofβ-1,3-glucanase gene with a cDNA length of 915bp, encoding 304 amino acids, obtained by RT-PCR combined with RACE technology, named as gsp (GenBank:FJ858156). Meanwhile, an ORF of 951bp cDNA ofβ-1,3-glucanase gene was cloned from the wild banana leaves at the normal temperature (28℃), encoding 316 amino acids, was named as gsp1 (GenBank:HQ018802). The sequences of gsp and gspl gene of the wild banana were significantly homologous with that of other banana varieties in GenBank. There was a 36bp deletion in the N-terminal of gsp in contrast with gspl. The results which were analyzed by bioinformatic methods showed that GSP and GSP1 belonged to glycosyl hydrolases family 17, which were basiCityβ-1,3-glucanase. The two proteins mainly located at cytoplasm and plasma membrane, respectively. GSP protein was a kind of hydrophilic protein, but GSP1 protein was the kind of hydrophobic protein. In contrast to gspl, gps had a 36bp deletion in N-terminal. Preliminarily, the gsp gene was thought to be an antifreeze-protein gene.6 DNA cloning of gsp and gspl genes of the wild banana from Sanming CityThe DNA sequences of gsp and gspl genes of the wild banana from Sanming City were cloned. The full sequences of gsp were 1540bp (GenBank:HQ018804), and the full sequences of gspl were 1531bp (GenBank:HQ018803). Sequence analyses indicated that gsp contained a 625bp intron, which splicing sites didn’t obey with the GT-AG rule. And gspl contained a 580bp intron, which splicing sites obeyed with the GT-AG rule. The differences of the intron between the two genes were most likely to the differences betweenβ-1,3-glucanase antifreeze protein gene and commonβ-1,3-glucanase gene.7 The expression ofβ-1,3-glucanase gene of the wild banana from Sanming City under cold stressUsing 18S rRNA as the reference gene, the transcriptional expression levels of gsp and gspl genes in the low-temperature treatment process were analyzed by q-PCR. The result showed that the highest expression of gsp gene of the wild banana was at 15℃treated 30h, the trend of changes was similar to Chi 12 gene of the wild banana. Though the expression of gspl gene varied unconspicuously under cold stress. All these showed that gsp and Chi 12 gene of the wild banana could respond to cold stress rapidly and had cumulative effect. The results in further explanation showed that gsp gene maybeβ-1,3-glucanase antifreeze protein gene.In summary, in the study the chitinase andβ-1,3-glucanase genes with characteristics of cold resistance were cloned from the wild banana from Sanming City under cold stress. Based on the molecular weight, structure and the expression under cold stress, Chi 12 and gsp were probably deduced to be antifreeze protein genes. This information would provide target gene for cold-resistant gene-engineering of banana and other tropical and subtropical crops. Simultaneously, the promoter sequences of Chi I land Chi 12 were cloned, which might lead to reveal the mechanism of cold-resistance and the expression and regulation of cold-resistance genes of the wild banana.更多还原