拟南芥psy基因cDNA的克隆及其植物表达载体的构建

为了获得胚乳组织特异性表达八氢番茄红素的转基因小麦,以拟南芥幼叶RNA为模板,由特异型引物通过RT-PCR一步法得到大小约为1.3kb的基因片段,将此片段连接在克隆载体pMD18-T进行测序,结果表明,该基因片段为八氢番茄红素合成酶基因(psy)cDNA片段。将psy基因片段正向插入植物表达载体pLRPT中高分子量麦谷蛋白亚基基因1Dx5启动子与nos终止子之间,pLRPT载体无1Dx5基因开放阅读框,运用菌落PCR对重组子进行筛选与鉴定,说明拟南芥psy基因已正确插入pL-RPT,成功构建了植物表达载体pLRPTPSY。     

小麦黄色素合成途径中Psy 基因的克隆及分子特性小麦黄色素合成途径中Psy 基因的克隆及分子特性

对普通小麦( Ttiticum aestivum) 黄色素(YP) 合成途径中的首要限速酶———八氢番茄红素合成酶(Psy) 基因进行克隆和测序, 并和玉米Psy 基因进行序列比对。结果表明, 在高和低YP 含量小麦品种中均扩增出一条长1 192 bp 的Psy 基因片段, 该片段包含一条可编码78 个氨基酸的小麦Psy 基因的外显子, 与玉米Psy 基因第4 外显子的核苷酸序列同源率为80 . 74% , 同源区域内有47 个SNPs , 但仅11 个SNPs 导致氨基酸编码序列的改变, 二者氨基酸序列的同源率达85 . 89% , 推测Psy 基因在不同物种中的表达具有较高的保守性。BLAST 聚类分析表明, 禾谷类植物Psy 基因的分类与物种的亲缘关系存在明显的相关性,小麦Psy 基因在系统进化中比禾谷类其他植物更为高级。     

小麦黄色素合成途径中Psy基因的克隆及分子特性

对普通小麦(Ttiticum aestivum)黄色素(YP)合成途径中的首要限速酶——八氢番茄红素合成酶(Psy)基因进行克隆和测序,并和玉米Psy基因进行序列比对。结果表明,在高和低YP含量小麦品种中均扩增出一条长1192bp的Psy基因片段,该片段包含一条可编码78个氨基酸的小麦Psy基因的外显子,与玉米Psy基因第4外显子的核苷酸序列同源率为80.74,同源区域内有47个SNPs,但仅11个SNPs导致氨基酸编码序列的改变,二者氨基酸序列的同源率达85.89,推测Psy基因在不同物种中的表达具有较高的保守性。BLAST聚类分析表明,禾谷类植物Psy基因的分类与物种的亲缘关系存在明显的相关性,小麦Psy基因在系统进化中比禾谷类其他植物更为高级。     

雨生红球藻八氢番茄红素合成酶基因的克隆及表征

雨生红球藻是一种单细胞绿藻,在多种逆境胁迫条件下能够大量合成并迅速积累虾青素,其积累量最高可达细胞干重的4%,从而成为目前最理想的天然虾青素合成工具.八氢番茄红素合成酶(PSY)是虾青素合成途径中第一个限速酶.分离了八氢番茄红素合成酶基因(psy)的全长cDNA及基因组DNA.其全长cDNA包括1200个碱基,编码400个氨基酸,基因组DNA包括5个外显子,4个内含子.系统发育分析结果显示,绿藻的八氢番茄红素合成酶基因形成一个进化枝,它们与高等植物的psy亲缘关系比较近.通过GenomeWalking的方法,分离了psy基因约1kb的5′侧翼序列.将含有TATA-box和CAAT-box的297bp的序列与LacZ报告基因构成嵌合的表达载体,用基因枪法转化雨生红球藻.lacZ的瞬间表达检测结果表明,这段上游序列能够驱动lacZ表达,具有启动子活性.     

小麦尿卟啉原Ⅲ合成酶基因克隆及序列分析

根据水稻已公布的尿卟啉原Ⅲ合成酶(UROS)基因和小麦EST的保守序列,设计特异性引物对小麦尿卟啉原Ⅲ合成酶基因的部分片段进行克隆,得到了364 bp的cDNA(命名为UROS1)。以UROS1作为种子进行电子克隆,得到一段长为1210 bp的cDNA序列,并设计特异性引物克隆到1个1077 bp cDNA序列。对该片段分析结果表明,克隆得到的小麦UROS基因包含了信号肽区和全长的成熟肽区。小麦UROS基因与水稻UROS基因的同源性为86%左右,其推导氨基酸序列与水稻和拟南芥蛋白序列同源性分别约91%和79%。动物、植物以及微生物间核酸序列的保守性较低,氨基酸序列保守性也不高,但都存在UROS保守结构域(Hem D)。进化分析显示,该酶在不同物种间的进化速度差异较大。     

八氢番茄红素合成酶(PSY)的生物信息学分析

目的:通过生物信息学方法对八氢番茄红素合成酶基因(PSY)及氨基酸序列分析,并构建三维结构。方法:运用生物信息学方法对八氢番茄红素合成酶基因及其蛋白质序列的理化性质、亲/疏水性、信号肽、跨膜结构域、糖基化位点,磷酸化位点,二级结构,功能结构域和三级结构进行预测分析。结果:PSY基因含1239bp的开放阅读框,编码氨基酸数为412,为碱性不稳定蛋白;八氢番茄红素合成酶富含Arg、Leu、Ala、Ser、Val等氨基酸,为亲水性蛋白质;PSY为非跨膜蛋白,不含信号肽,具有多个磷酸化位点,α螺旋和无规卷曲是其主要结构元件。结论:用同源建模的方法构建其三维结构,得到合理模型,为采用生物工程提高番茄红素产量提供理论依据。     

果实特异性RNAi介导的Lcy基因沉默来增加番茄中番茄红素的含量

根据GenBank中番茄的番茄红素β-环化酶(Lcy)基因序列和八氢番茄红素去饱和酶基因(Pds)启动子序列设计特异引物从番茄基因组DNA中分别扩增出了Lcy基因的高度保守的长302bp的DNA片段和长1790的Pds启动子片段。根据RNAi的原理,将Lcy基因的DNA片段以正反两个方向通过一段内含子序列连接在一起形成RNAi片段,将该片段与Pds启动子一起插入到pVCT2020的表达载体中,通过农杆菌介导的方法转化番茄,获得转基因植株5棵,PCR检测证实外源片段已成功导入番茄基因组中。收获转色期后20d左右的完全成熟的番茄果实提取番茄红素进行含量分析,结果显示转基因番茄果实中番茄红素的含量极大的增加了。上述结果表明通过RNAi果实特异性的抑制类胡萝卜素代谢途径中生物合成酶基因的表达能够极大的增加番茄果实中番茄红素的含量。这为通过基因工程手段提高番茄果实中的营养价值提供了参考。     

栀子八氢番茄红素合成酶基因的分离及表达分析

为探讨栀子(Gardenia jasminoides)果实中藏花素的合成机理,克隆了栀子类胡萝卜素生物合成的关键酶八氢番茄红素合成酶(GjPSY)基因的全长cDNA。结果表明,推导的GjPSY氨基酸序列与双子叶植物来源的GjPSY亲缘关系较近。采用HPLC检测栀子果实中的藏花素-1含量为(3.96±1.48) mg g-1,在叶片中未检出。通过RT-PCR分析表明,GjPSY在栀子叶片和果实中均有表达,且表达水平一致。因此推测,GjPSY的转录水平与果实中藏花素-1的合成无关。     

草莓MDHAR及AO基因的克隆及序列分析

从新鲜幼嫩‘丰香’草莓(Fragaria×ananassa cv.Toyonaka)果实中提取分离总RNA,反转录成cDNA,根据已报道的其他植物单脱氢抗坏血酸还原酶(MDHAR)及抗坏血酸氧化酶(AO)基因的保守区分别设计 一对引物,通过PCR扩增均得到目的条带.序列分析发现:mdhar基因片段长372bp,与刺梨同源性最高达96％,该片段编码123个氨基酸,推导的氨基酸序列与苹果属植物同源性为91％,与其他植物该基因编码的氨基酸序列也有较高相似性;a基因片段长842 bp,编码280个氨基酸,该片段与其他多种植物的ao基因均具有较高同源性,与甜瓜和黄瓜ao基因的同源性最高,均为70％,编码的氨基酸序列与其他多种植物均具有70％左右的相似性.     

大豆GmPDS基因的克隆及VIGS表达载体构建和鉴定

病毒诱导的基因沉默(virus induced gene silencing,VIGS)技术是近年来发展起来的一种反向遗传学快速研究基因功能的方法,对于植物特别是难于转化的大豆而言尤其适用。本研究采用PCR技术从大豆(Glycine max)基因组中克隆了八氢番茄红素去饱和酶(phytoene desaturase,PDS)基因的部分序列,命名为GmPDS(Glycinemax PDS)。该片段长430bp,序列分析表明,该基因与大豆八氢番茄红素去饱和酶(GenBank登录号:M64704)cDNA序列同源性为99%。双酶切GmPDS片段和烟草脆裂病毒载体(pTRV2),构建了重组载体pTRV2-GmPDS,并将该重组载体分别转化农杆菌C58C1/pMP90、GV3101和LBA4404,为分析pTRV载体是否可以浸染大豆奠定了基础。     

梭梭Na+/H+逆向转运蛋白基因克隆及分析

采用RT-PCR、RACE方法从超旱生、耐盐植物梭梭中扩增出Na+/H+逆向转运蛋白基因的开放阅读框架,其核苷酸序列长1 683bp,推测的氨基酸序列全长为560个氨基酸残基。含有多个物种Na+/H+逆向转运蛋白基因的高度保守序列氨氯砒嗪脒的结合位点(LFFIYLIPPI)。序列一致性分析结果显示,该cDNA片段与同科植物NHX基因的一致性为70%～80%,但与不同科植物的一致性较低,仅为60%,表明该基因在进化上存在多样性,但它们都具有氨氯砒嗪脒结合位点,对Na+具有高度专一性,对植物的耐盐性起着重要作用。     

棉蚜2个乙酰胆碱酯酶cDNA片段的基因克隆与序列分析

采用RT－PCR技术,利用简并引物从棉蚜（Aphis gossypii Glover)中克隆出2个乙酰胆碱酯酶基因的cDNA片段,Ag,ace 1l和Ag.ace2.Ag.ace1基因的cDNA片段为282bp,编码94个氨基酸;Ag.ace2基因的cDNA片段为264bp,编码88个氨基酸。扩增获得的2个乙酰胆碱酯酶基因cDNA片段所编码的氨基酸序列均与其他昆虫的乙酰胆碱酯酶基因有很高的同源性。首次从一种昆虫中克隆出2个乙酰胆碱酯酶基因片段,为同一种昆虫中存在多个乙酰胆碱酯酶基因的假设提供了直接的分子生物学证据。     

Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects

The nucleotide sequence coding for the fourth component of mouse complement (C4) has been determined from a cloned genomic DNA fragment and a cloned cDNA fragment. The amino acid sequence of the protein was deduced. The single chain precursor protein (pro-C4) consists of 1719 amino acid residues. The mature beta, alpha, and gamma subunits contain 654, 766, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted for the beta chain, three for the alpha chain, and none for the gamma chain. From a comparison with human C4 cDNA sequence an extensive overall sequence homology, 79% in nucleotides and 76% in amino acids, is observed. There is conservation in both the position and number of cysteine residues in human and mouse C4. We compared the mouse C4 amino acid sequences with those of mouse C3 and human alpha 2-macroglobulin and the evolutionary relationship among these three proteins is discussed.     

Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K

A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.     

番杏Actin基因片段的克隆及生物信息学分析

本实验为研究番杏(Tetragonia tetragonioides)功能基因表达模式提供内参基因,根据登录在NCBI上植物的Actin基因的保守区域设计简并性引物,通过RT-PCR克隆获得番杏的Actin基因片段,将该片段连接于载体pGEM-T后进行测序,并将该基因序列通过生物信息学软件进行分析。结果显示,克隆所得基因片段大小为598 bp,编码198个氨基酸;该序列与登录在NCBI上的其他植物的Actin基因的核苷酸序列的同源性最大可达86%以上,编码蛋白的氨基酸序列同源性在88%以上。结果表明,本研究克隆所得的基因序列为番杏Actin基因片段。该基因命名为TtActin1,在Gen Bank的登录号为MH33308。     

Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina

A cDNA encoding a nicotinamide adenine dinucleotide (NAD+) -dependent glycerol 3-phosphate dehydrogenase (GPDH) has been cloned by rapid amplification of cDNA ends from Dunaliella salina. The cDNA is 3032 base pairs long with an open reading frame encoding a polypeptide of 701 amino acids. The polypeptide shows high homology with published NAD+ -dependent GPDHs and has at its N-terminal a chloroplast targeting sequence. RNA gel blot analysis was performed to study GPDH gene expression under different conditions, and changes of the glycerol content were monitored. The results indicate that the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis. The 701-amino-acid polypeptide is about 300 amino acids longer than previously reported plant NAD+ -dependent GPDHs. This 300-amino-acid fragment has a phosphoserine phosphatase domain. We suggest that the phosphoserine phosphatase domain functions as glycerol 3-phosphatase and that, consequently, NAD+ -dependent GPDH from D. salina can catalyze the step from dihydroxyacetone phosphate to glycerol directly. This is unique and a possible explanation for the fast glycerol synthesis found in D. salina.     

Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana.

A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene. A fragment of the predicted size was amplified from genomic DNA from Arabidopsis thaliana. Overlapping cDNA clones, some with homology to the PCR amplified fragment, were identified and sequenced. The assembled nucleic acid sequence is 4720 bp and encodes a protein of 1534 amino acids which has significant homology to prokaryote and mammalian cytosine methyltransferases. Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain. The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytosine-5 methyltransferases and shows 50% homology to the murine enzyme in the methyltransferase domain. The amino terminal domain is only 24% homologous to the murine enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man. In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana.