CAJ | 학술논문

利用RT-PCR方法从大豆根部组织获得Glyma03g34310.1开放阅读框(ORF)全长,经测序验证、Blast比对与同源性分析发现该序列编码的蛋白质与其他植物的 TIP1;1蛋白具有较高的相似性,故命名为 GmTIP1;1基因(GenBank登录号为AK285481),该基因ORF长753 bp,编码1个包含250个氨基酸的蛋白,在ORF内部第381个核苷酸处含有1个94 bp的内含子,符合↓GT--AG↓的剪接方式;系统进化树分析发现GmTIP1;1聚类到豆科植物分支,其他不同科的植物也有规律地聚到了不同分支,推测该蛋白氨基酸序列可以作为植物分类的依据之一;半定量RT-PCR结果表明该基因在大豆的不同器官、不同器官的不同发育阶段均具较高且同等的表达水平,暗示该基因在植物的整个发育进程中均具重要作用;在盐胁迫的不同时间点其表达量有下降的趋势,但仍然保持较高的表达水平;以pYES2为酵母表达载体,转化酿酒酵母INVSc1菌株,获得重组酵母INVSc1(pYES2-GmTIP1;1),转化菌株在盐胁迫下的存活率明显高于对照INVSc1(pYES2),而在干旱胁迫下则没有显著差异,表明该基因的表达能有效地提高酵母的耐盐性.
이용RT-PCR방법종대두근부조직획득Glyma03g34310.1개방열독광(ORF)전장,경측서험증、Blast비대여동원성분석발현해서렬편마적단백질여기타식물적 TIP1;1단백구유교고적상사성,고명명위 GmTIP1;1기인(GenBank등록호위AK285481),해기인ORF장753 bp,편마1개포함250개안기산적단백,재ORF내부제381개핵감산처함유1개94 bp적내함자,부합↓GT--AG↓적전접방식;계통진화수분석발현GmTIP1;1취류도두과식물분지,기타불동과적식물야유규률지취도료불동분지,추측해단백안기산서렬가이작위식물분류적의거지일;반정량RT-PCR결과표명해기인재대두적불동기관、불동기관적불동발육계단균구교고차동등적표체수평,암시해기인재식물적정개발육진정중균구중요작용;재염협박적불동시간점기표체량유하강적추세,단잉연보지교고적표체수평;이pYES2위효모표체재체,전화양주효모INVSc1균주,획득중조효모INVSc1(pYES2-GmTIP1;1),전화균주재염협박하적존활솔명현고우대조INVSc1(pYES2),이재간한협박하칙몰유현저차이,표명해기인적표체능유효지제고효모적내염성.
The full length open reading frame (ORF) of gene named Glyma03g34310.1 was amplified from the soybean root tis-sues by reverse transcriptase polymerase chain reaction (RT-PCR) method. Sequencing, Blast and homology analyses showed that the amino acids encoded by this gene had the higher similarity with the TIP1;1 from other species, so designated GmTIP1;1 (GenBank accession number:AK285481), its ORF was 753 bp, encoded a polypeptide with 250 amino acids, and contained a 94 bp intron at the site of the 381th nucleotide complying with the↓GT--AG↓mode of splicing. GmTIP1;1 had two copies in the soybean genome, with the other one of Glyma19g37000.1. Multiple alignment using protein indicated that GmTIP1;1 contained six conserved transmembrane domains and two higher conserved NPA motifs. Phylogenetic tree analysis using MEGA5.05 showed that GmTIP1;1 was indeed grouped into the legumes clade and several different clades which belonged to the different plant coleus were regularly separated based on the TIP1;1 protein sequences, which implied that TIP1;1 sequence probably could be regard as a proof of plant taxonomy. Semi-quantity RT-PCR analysis demonstrated that the GmTIP1;1 gene constitutively ex-pressed in soybean organs including root, stem, leaf, flower and pod, and the expression levels were no obvious difference in dif-ferent tissues at the different developmental stages, which implied GmTIP1;1 gene plays important roles in plant growth. The expression of GmTIP1;1 appeared a declined trend at the different time points under the treatment of salt solution (200 mmol L–1 NaCl), although still showing the abundant transcript. In addition, the recombinant plasmid pYES2-GmTIP1;1 was constructed by inserting the GmTIP1;1 gene into the yeast expression vector pYES2. The recombinant plasmid pYES2-GmTIP1;1 was trans-formed into yeast Saccharomyces cerevisiae INVScl, then treated with salt and drought stresses, respectively. The results showed that the survival rate of the recombinant yeast INVScl (pYES2-GmTIP1;1) was higher than that of the control strain under salinity condition, but no difference under drought condition. These results indicated that the heterologous expression of GmTIP1;1 could effectively improve the tolerance of yeast to salinity stress.