梨小食心虫几丁质合成酶1基因的克隆与表达分析

【目的】克隆梨小食心虫Grapholitha molesta(Busck)几丁质合成酶1基因,分析该基因的分子特征及时空表达模式,为探析其生理功能奠定基础。【方法】利用简并引物和RACE技术从梨小食心虫5龄幼虫和蛹中克隆几丁质合成酶1基因的全长c DNA序列,同时获得其两个可变剪切外显子序列,并用邻接法(neighbor-joining method)与其他昆虫同源序列构建系统进化树。利用RTq PCR技术研究该基因及两个可变剪切外显子在1日龄预蛹不同组织(头、体壁、脂肪体、中肠、气管和马氏管)中以及不同发育阶段(2-5龄幼虫、预蛹、蛹和成虫)的表达特性。【结果】克隆获得梨小食心虫几丁质合成酶1基因,将其命名为Gm CHS1,该基因编码1 565个氨基酸,包含了16个跨膜螺旋,两个可变剪切外显子包含177个碱基,编码59个氨基酸序列,分别命名为Gm CHS1a(Gen Bank登录号:MF000781)和Gm CHS1b(Gen Bank登录号:MF000782)。系统发育树同源分析结果表明,Gm CHS1属于几丁质合成酶1,Gm CHS1a和Gm CHS1b分别归属于可变剪切外显子CHS1a和CHS1b。组织表达模式表明,Gm CHS1基因在体壁中表达量最高,其次是在头和气管中,其余组织中表达量较低或不表达;发育表达模式表明,该基因在各个发育阶段均有表达,幼虫蜕皮期、预蛹-蛹和蛹-成虫转变过程中表达量上调。Gm CHS1a在体壁和头部的表达量高于Gm CHS1b,而在气管和脂肪体中的表达量略低于Gm CHS1b,两者在中肠和马氏管中表达量都很低。在梨小食心虫不同发育阶段,Gm CHS1a的表达趋势表现为在幼虫蜕皮和预蛹-蛹转变过程中高表达;Gm CHS1b在幼虫各个阶段表达量都较低,在预蛹-蛹和蛹-成虫转变过程中高表达。【结论】Gm CHS1a和Gm CHS1b属于昆虫几丁质合成酶1家族,其基因在梨小食心虫不同组织及发育阶段的表达量显著不同,推测其在梨小食心虫发育过程中发挥着不同的作用。本研究为进一步探索该基因在梨小食心虫体内的功能奠定了基础。     

百合查尔酮合成酶基因的克隆与分析

以西伯利亚百合为试材,通过半巢式PCR和RT-PCR技术分别克隆了查尔酮合成酶基因(CHS)的DNA和cDNA.生物信息学分析显示,CHS的DNA序列全长1 397 bp(登录号HM622754),包含2个外显子和1个内含子;cDNA序列编码区全长1 182 bp(登录号HQ161731),编码393个氨基酸,具有3个典型的CHS蛋白结构域:N-末端结构域(Lys3-Pro229)、C-末端结构域(Gln239-Pro389)和聚合酶Ⅲ结构域(Met1-Thr391);不同百合品种的CHS基因编码的氨基酸序列相似性高达98%,表明百合CHS基因在进化上呈现出十分保守的趋势;不同植物CHS基因序列的系统进化邻接树结果表明:百合与单子叶植物鸢尾及禾本科的水稻、大麦、玉米等亲缘关系更为接近.     

梅花类黄酮3'-羟化酶基因片段基于基因组DNA的简并PCR法克隆

用本研究设计的"预先去杂-SDS法"从梅花嫩叶提取到高质量的基因组DNA.根据11条已公开发表的并提交到GenBank的类黄酮3'-羟化酶基因cDNA的假定氨基酸序列的保守区设计2个正向简并引物和3个反向简并引物组成6对引物,仅有1对引物能以PCR法同时从梅花'南京红须'、'南京红'和'粉皮宫粉'的基因组DNA扩增到一个469 bp的核苷酸片段,这3个片段在总体上有99.72%的一致性,与11条类黄酮3'-羟化酶基因cDNA的相应区域有65.57%的一致性.同时,"GGEK"并非类黄酮3'-羟化酶的特征性模体.这是首次从木本植物的基因组DNA克隆到类黄酮3'-羟化酶基因片段.本研究结果可为梅花类黄酮3'-羟化酶基因全长的克隆奠定基础.     

蓝粒小麦花青素合成途径中的查尔酮合酶基因和类黄酮3′5′-羟化酶基因3′末端的克隆和表达分析

采用RT-PCR和RACE方法从蓝粒小麦(Triticum aestivum L.×Thinopyrum ponticum(Podp.)Z.W.Liu et R.R.-C. Wang)发育种子中克隆到两个查尔酮合酶基因(TaCHS.t1,TaCHS.w1),分别编码394个氨基酸,二者核苷酸和氨基酸序列的同源性分别为96.0%和98.9%;而且克隆到一个类黄酮3′5′-羟化酶基因(F3′5′H)3′-末端.分别从长穗偃麦草(Thinopyrum ponticum(Podp.)Z.W.Liu et R.R.-C.Wang)、蓝粒小麦、白粒小麦和中国春基因组中分离到查尔酮合酶基因(CHS)的全长序列(ThpCHS.tg,TaCHS.bg,TaCHS.wg,TaCHS.csg),它们的核苷酸序列之间具有很高的同源性,且均含有一个内含子(intron),序列差异主要在内含子.通过DNA序列比较,发现一个CHS与亲本之一的长穗偃麦草基因组同源性达100%,一个CHS与另一白粒亲本基因组同源性达99%,表明来自于父、母本的CHS在蓝粒发育的种子中均表达.Southern杂交结果表明CHS在小麦中的拷贝数至少有4个,不同颜色的蓝粒小麦、白粒小麦间拷贝数基本一致,但都与长穗偃麦草有差异,根据以上结果初步判定CHS在蓝粒小麦中属于一个多基因家族.Reverse Northern分析表明CHS在开花后15 d发育的蓝粒小麦中具有较强的表达;花后21 d F3′5′H和DFR的转录本积累显著高于CHS,基本上检测不到CHS的表达.这三个基因在蓝粒小麦中的表达顺序与其他植物花青素合成途径中的基因表达次序一致:CHS先于F3′5′H,F3′5′H先于DFR.实验还表明F3′5′H和DFR在幼叶中表达也较强,CHS仅在发育种子中表达,具有组织特异性.花后21 d,F3′5′H在白粒、蓝粒小麦和中国春种子中均强烈表达,蓝粒小麦CHS和DFR转录本比白粒小麦多.因此,认为在蓝粒小麦中存在花青素生物合成途径,在蓝色色素形成过程中,该途径中的结构基因受到调节基因的调控.     

两个大豆GmSBP基因的特征、亚细胞定位及对非生物胁迫的响应

我国南方春大豆种子发育过程中,常处于高温、高湿季节,加之种子本身富含蛋白(约40%)和脂肪(约20%),导致南方春大豆种子易劣变。本项目组前期差异蛋白质组学研究发现蔗糖结合蛋白在高温高湿胁迫168 h时在种子田间劣变抗性品种湘豆3号发育种子中呈下调表达。为进一步从分子水平了解Gm SBP基因表达以及响应高温高湿胁迫的特性,本研究利用RT-PCR技术从大豆扩增出两个Gm SBP基因(Gm SBP2和Gm SBPL)。两个基因编码的蛋白均为亲水性,不完整的膜蛋白。荧光定量PCR分析表明:在高温高湿条件下,种子田间劣变不抗品种宁镇1号和抗性品种湘豆3号发育种子中Gm SBP2和Gm SBPL基因表达量均受高温高湿胁迫影响,也会导致种子中蔗糖和可溶性糖含量变化。在籽粒发育过程中,Gm SBP2和Gm SBPL基因表达量在花后30 d左右达到最高,对应时期的蔗糖和可溶性糖含量也达到最大值。组织特异性显示Gm SBP和Gm SBPL基因在不同组织间存在差异表达。亚细胞定位结果表明Gm SBP2和Gm SBPL蛋白均定位在细胞膜和细胞质中。以上结果表明Gm SBP2和Gm SBPL基因可能参与了植物非生物胁迫的应答过程,这将从一个侧面丰富我们对大豆种子田间劣变性和劣变抗性的认识。     

梅花类黄酮3′-羟化酶基因片段基于基因组DNA的简并PCR法克隆(英文)

用本研究设计的“预先去杂—SDS法”从梅花嫩叶提取到高质量的基因组DNA。根据11条已公开发表的并提交到GenBank的类黄酮3′-羟化酶基因cDNA的假定氨基酸序列的保守区设计2个正向简并引物和3个反向简并引物组成6对引物,仅有1对引物能以PCR法同时从梅花‘南京红须’、‘南京红’和‘粉皮宫粉’的基因组DNA扩增到一个469 bp的核苷酸片段,这3个片段在总体上有99 .72 %的一致性,与11条类黄酮3′-羟化酶基因cDNA的相应区域有65 .57 %的一致性。同时,“GGEK”并非类黄酮3′-羟化酶的特征性模体。这是首次从木本植物的基因组DNA克隆到类黄酮3′-羟化酶基因片段。本研究结果可为梅花类黄酮3′-羟化酶基因全长的克隆奠定基础。     

水蕨(Ceratopteris thalictroides)查尔酮合成酶基因的克隆和表达（英文）

查尔酮合酶(chalcone synthase,CHS)是植物类黄酮化合物合成的关键酶,有关蕨类植物CHS基因的序列及功能信息尚不完善。本研究采用快速扩增c DNA末端(RACE)技术克隆获得了模式蕨类植物——水蕨(Ceratopteris thalictroides)Ct CHS基因(Gen Bank登录号:JX027616.1),其c DNA序列全长为1616 bp,具有3个外显子和2个内含子,开放阅读框(ORF)为1215 bp,编码404个氨基酸。进化树分析表明,Ct CHS与问荆(Equisetum arvense)、松叶蕨(Psilotum nudum)和3种薄囊蕨的查尔酮合成酶基因聚为一枝,说明这些蕨类植物亲缘关系较近且为单系起源。通过构建原核表达体系成功获得Ct CHS蛋白的多克隆抗体并用于免疫印迹分析,结果表明Ct CHS基因的表达明显受紫外光(UV)诱导。Ct CHS基因的克隆与表达分析为进一步研究水蕨类黄酮化合物的合成及其调控机制提供了依据。     

大豆再生相关基因GmBIN2的克隆及生物信息学分析

根据前期实验获得的大豆Gm BIN2基因登录号,从大豆中克隆Gm BIN2基因的全长CDS序列,得到大豆Gm BIN2基因。对大豆再生相关基因Gm BIN2的启动子序列、氨基酸序列、编码的蛋白质结构、亲疏水性以及同源进化树进行分析,结果表明,大豆再生相关基因Gm BIN2编码区c DNA长度为1 125 bp,编码374个氨基酸,Gm BIN2编码的蛋白为亲水性蛋白;分析其蛋白功能结构域发现,Gm BIN2蛋白具有丝氨酸/苏氨酸激酶催化域,为PKc-like超家族成员;构建系统进化树发现其与野生大豆亲缘较近。本研究的实验结果有利于更加深入的研究Gm BIN2基因在大豆再生过程中的关键作用,为提高大豆再生效率提供依据。     

苦荞和甜荞查尔酮合成酶基因的克隆及序列比较

以苦荞品种‘西农9920’和甜荞品种‘西农9976’为材料,根据其它植物查尔酮合成酶(chalcone synthase,CHS)基因DNA序列的保守区域设计的一对简并引物,进行PCR扩增,从2种荞麦基因组中克隆出了长度均为860 bp的CHS基因片段,对其进行回收、克隆,挑选阳性克隆测序;序列分析表明这2个片段含有CHS基因的N端和C端的结构域,分别为苦荞和甜荞的CHS基因片段,命名为FtCHS和FeCHS。对获得的2种荞麦CHS基因的DNA序列进行比较分析,发现两者间存在多达43处单碱基多态性,这些单碱基多态性可能是苦荞和甜荞种子中类黄酮含量差异的重要原因之一。苦荞和甜荞CHS与其它植物CHS的氨基酸序列的进化分析表明,其与同为蓼科的掌叶大黄和石竹科的满天星的同源性较近。     

石榴果色合成相关基因CHS和CHI的表达特性分析

花色苷是类黄酮家族中重要的一类次生代谢产物,对果实呈色起重要作用。CHS (查尔酮合成酶)和CHI (查尔酮异构酶)为花色苷合成提供了前体物质,是花色苷合成所不可或缺的。利用RT-PCR和RACE方法,本研究从石榴果皮中克隆了与花色苷合成相关的CHS基因和CHI基因的cDNA全长,同时采用qRT-PCR研究了这两个基因在三个不同色泽石榴品种‘红宝石’、‘水晶甜’、‘墨石榴’发育期内的表达模式,并分析了果皮花色苷含量变化与基因转录水平的关系。结果表明,石榴中CHS和CHI基因cDNA全长分别为1 197 bp和693 bp,分别编码398和230个氨基酸,命名为PgCHS和PgCHI,在GenBank中的登录号分别为KF841615和KF841616。在氨基酸水平上,Pg CHS与荔枝、葡萄、山竹等果树的同源性达到90%以上。Pg CHI与果树中龙眼、梨、美洲葡萄、桑树等同源性达到70%以上。qRT-PCR结果显示,CHS和CHI基因的表达模式随色泽发育期和品种不同而有差异。在‘红宝石’石榴中,该两个基因都有前期和后期两个表达高峰期;而‘水晶甜’石榴中这两个基因的表达高峰期均出现在中后期;‘墨石榴’发育初期时CHS和CHI的表达量最高,以后的表达量都较低。同一品种内,CHS和CHI的表达具有协同性,两者的协同性表达有利于花色苷及其他类黄酮相关产物的合成。3个品种中CHS和CHI基因的表达与花色苷的积累并不一致。     

Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica)

We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down‐regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down‐regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS‐silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS‐silenced line was smaller than the ‘Royal Gala’ controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS‐silenced line compared with the ‘Royal Gala’ control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development.     

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits

Flavonoids possess diverse health‐promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone‐3‐hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm‐specific GluB‐1 promoter or embryo‐ and aleurone‐specific 18‐kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB‐II‐type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.     

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

CHS silencing suggests a negative cross-talk between wax and flavonoid pathways in tomato fruit cuticle

Tomato fruits (Solanum lycopersicum L.) accumulate flavonoids in their cuticle and epidermal cells during ripening. These flavonoids come from de novo biosynthesis due to a significant increase in chalcone synthase (CHS) activity during ripening. Virus-induced gene silencing (VIGS) of tomato fruits have been used to down-regulate SlCHS expression during ripening and analyze the effects at the epidermal and cuticle level. Besides the expected change in fruit color due to a lack of flavonoids incorporated to the cuticle, several other modifications such as a decrease in the amount of cutin and polysaccharides were observed. These indicate a role for either flavonoids or CHS in the alteration of the expression levels of some genes involved in cuticle biosynthesis. Moreover, a negative interaction between the 2 cuticle components, flavonoids and waxes, suggests a relationship between these 2 metabolic pathways.