水稻Ds插入具芒突变体相关基因的功能预测

[目的]研究水稻Ds插入具芒突变体形成的分子机理。[方法]采用TAIL-PCR技术,从水稻Ds插入具芒突变体中克隆出Ds侧翼基因序列,分析被插入基因的结构,并分析预测基因编码的蛋白功能。[结果]Ds插入在具芒突变体7号染色体Os07g0588700基因前1 339 bp处。Ds插入位置的下游基因编码产物含一个锌指区CX2CX3FX5LX2HX3H,且含高度保守的QALGGH保守区,为水稻的单锌指蛋白。[结论]Ds转座元件插入基因组中,影响了编码锌指蛋白基因的表达调控,使突变体显示出具芒的表型。     

RBSDV-S10基因和SCMV-CP基因双价植物表达载体的构建

克隆了水稻黑条矮缩病毒（RBSDV）的外壳蛋白S10基因的片段和甘蔗花叶病毒（SCMV）的外壳蛋白CP基因的片段,将两个片段在pMD18-T Simple Vector上连接起来。获得的S10-CP片段全长为830bp,将其分别以正向和反向插入植物表达载体pMCG161,构建了含两种不同病毒来源基因的RNAi植物表达载体pMCG161-CP10,为进一步转化玉米,探索利用RNAi技术同时防治玉米矮花叶病和玉米粗缩病奠定了基础。     

Inserted sequences in bovine satellite DNA's

The nucleotide sequence of the 1413-base-pair repeat unit of bovine 1.711a satellite DNA (density in cesium chloride, 1.711 grams per cubic centimeter) has been determined. The repeat unit contains two segments consisting of variants of a basic 23-base-pair sequence that is closely related to sequences of bovine 1.706 satellite DNA. A third segment of the repeat unit contains an unrelated 611-base-pair sequence that is not internally repetitive. This segment is flanked by inverted repeats of 8 base pairs and, on one side, by a direct repeat of the terminal sequence. A related segment is present in bovine 1.711b satellite DNA and is inserted into sequences derived from the 1.715 satellite. These nucleotide sequences suggest the timing of some of the stages in the evolution of these complex, closely related satellite DNA's and indicate the mechanisms inherent in their divergence from a common ancestor.     

A single genetic unit specifies two transposition functions in the maize element activator

The self-mobile maize transposable element Ac (Activator) displays two trans-acting genetic functions: it induces transposition of the element Ds (Dissociation) but, as its dosage is increased, it also inhibits transposition. Previous work has shown that the 4563 base pair (bp)-long Ac element contains three open reading frames (ORF's) and that a deletion in ORF 1 in wx-m9(Ds), a Ds derivative from Ac isolated at the wx (waxy) locus, results in loss of transposition. The Ds element in the bronze allele bz-m2(DI) is shown to have arisen from Ac by a 1312-bp deletion that is located almost entirely within ORF 2 and does not affect ORF 1. The Ds elements in wx-m9(Ds) and bzm2(DI), defective in ORF 1 and ORF 2, respectively, do not complement genetically to restore the transposition function of Ac; therefore, this function must be specified jointly by ORF's 1 and 2. Furthermore, since bz-m2(DI) does not contribute to Ac's inhibitory dosage effect, both Ac properties result from the expression of the same genetic functional unit.     

Waxy allele diversity in waxy maize landraces of Yunnan Province,China

Waxy maize is one of the main fresh-eating maize types, and a mutation of the waxy gene causes the waxy character of maize grains.  China is rich in waxy maize landraces, and Yunnan and its surrounding areas, are the place of origin and genetic diversity center of Chinese waxy maize.  The six known waxy alleles of Chinese waxy maize are wx-D7, wx-D10, wx-Cin4, wx-124, wx-Reina, and wx-Xuanwei.  The mutation sites of these alleles all occur in the coding region of the waxy gene, however, the mechanism by which the waxy characteristic is caused by the mutation in the regulatory region has only been reported rarely in maize.  In this study, 405 waxy maize landraces from Yunnan were used as materials to identify the insertion and deletion of a large sequence fragment in the upstream ~3.5 kb regulatory region of the waxy gene by molecular marker detection.  Three different waxy alleles were identifed in this study: wx-PIF/Harbinger, wx-hAT and wxElote2.  These three types of mutations all represented transposons inserted into the regulatory region of the waxy gene.  Wx-PIF/Harbinger was a 304-bp MITE class transposon insertion belonging to the PIF/Harbinger family, while wx-hAT was a 560-bp MITE class transposon insertion belonging to the hAT family, and wx-Elote2 was a 6 560-bp LTR-like transposon insertion.  In this study, the alleles were identifed for more than 70% of the waxy maize landraces in Yunnan, which provids a basis for the utilization of these waxy maize landraces.     

Single-copy inverted repeats associated with regional genetic duplications in gamma fibrinogen and immunoglobulin genes

We have found that a portion (150 base pairs) of the seventh exon of the human gamma fibrinogen gene is duplicated in the preceding intron. This duplicated sequence, termed a "pseudoexon," is flanked on each side by a single-copy inverted repeat sequence consisting of 102 base pairs. Frequencies of point substitutions indicate that both the pseudoexon and the inverted repeat sequence arose approximately 10 to 20 million years ago. The generality of this type of duplication is suggested by the occurrence of a similar duplication in the mouse immunoglobulin mu-delta region. As in the fibrinogen pseudoexon, the portion of the immunoglobulin mu-delta region containing the duplication and the inverted repeat was reported to be single-copy in the mouse genome. Since both of the first two single-copy inverted repeats to be sequenced are associated with regional duplications, it is likely that many of the single-copy inverted repeat sequences, which make up 1 to 2 percent of the genome, are also associated with regional duplications.     

CRISPRs在德氏乳杆菌中的检测与同源性分析

本研究检测了本实验室德氏乳杆菌中的CRISPRs结构,并对其重复序列和间区序列进行了同源性分析.结果显示,供试菌株L7和L9中均含有CRISPRs序列,分别为3 323 bp和3 015 bp,多重比对后的重复序列8～12位与21～17位碱基互补形成回文结构;重复序列大小(29 bp)与标准菌株基本相同,但其同源性与菌...     

转基因油菜W-4 T-DNA旁侧序列分析与事件特异性检测

W-4是通过农杆菌介导法将fad2基因的反向重复序列表达框转入甘蓝型油菜Westar后获得的转基因高油酸油菜品系.为建立W-4的转基因事件特异性PCR检测技术,应用温度不对称PCR（TAIL-PCR）扩增获得转基因油菜W-4的T-DNA插入位点的左、右旁侧序列.其中右边界旁侧序列长度为290 bp,其碱基组成G＋C含量为31.27％、A＋T含量为68.73％;左边界旁侧序列长度为365 bp,其碱基组成G＋C含量为32.6％、A＋T含量为67.4％,表明该T-DNA整合在富含AT区.序列比对结果发现,该转基因事件中,T-DNA左边界序列完全整合到油菜基因组中,仅有1个碱基由G转换成了A.而右边界则缺失了包括RBborder在内的62个碱基.结果表明：转基因高油酸油菜T-DNA的整合是一次无载体序列的整合.依据左、右边界旁侧序列和转基因载体的T-DNA左右边界序列设计了2对特异性引物TLF/TLR和TRF/TRR,能从W-4基因组DNA中扩增出大小分别为485和405 bp的预期产物,而在其他转基因油菜、非转基因油菜的基因组DNA和空白对照中均无特异性扩增产物,据此建立了W-4的转基因事件特异性PCR检测技术.应用该检测技术可以从含有0.1％ W-4基因组DNA的混合样品中扩增出特异产物,检测灵敏度达0.1％.可对W-4的转基因事件进行特异性检测.     

鸡MHC-B区域内复合微卫星LEI0258位点等位基因的序列变异

在对15个样品鸡MHC-B区域内复合微卫星位点LEI0258的等位基因进行基因型分析的基础上,进一步开展了等位基因PCR产物的直接测序或克隆测序,并将获得的序列与GenBank中的18条同源序列作了比较.结果表明:LEI0258位点等位基因片段大小分别为247bp、249bp、307bp、357bp、369bp、381bp、393bp或443bp的等位基因具有不同的DNA序列或结构;上、下游共有3个in/dels和7个SNPs;由12bp和13bp组成的重复单元中也存在一定的变异,其中,HRJ23中一个13bp的重复单元由标准的"CTATGTCTTCTTT"变为"CTATATCTTCTTT",HRJ5和JT01中各有一个12bp的重复单元由标准的"CTTTCCTTCTTT"变为"CTTC-CCTTCTTT",而HRJ5、HRJ15和P22中也各有一个12bp的重复单元由标准的"CTTTCCTTCTTT"变为"CTTTTCTTCTTT".     

Isolating the Mutator Transposable Element Insertional Mutant Gene mio16 of Maize Using Double Selected Amplification of Insertion Flanking Fragments (DSAIFF)

Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (mio16) was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments (DSAIFF), was employed to isolate the Mu flanking fragments (MFFs) of mio16. The target site duplications (TSDs) isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and mio16 was mapped in silico onto the physical position ranged from 229 965 021 to 229 965 409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of mio16 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of mio16 is alternated by Mu insertion.     

AtPIP5K2基因参与拟南芥盐胁迫的调节过程

植物在长期进化过程中演化出不同机制来适应环境中的各种胁迫,如盐碱、干旱等.该研究从拟南芥T-DNA插入突变体库中筛选到一个对盐反应不敏感的突变株系eto(enhanced tolerance to osmotic stress),种子萌发和幼苗生长试验表明eto突变株系早期生长发育对盐胁迫不敏感.TAIL-PCR分析表明eto突变株系中T DNA插入在拟南芥1号染色体上(BAC F3M18的27502位置),位于拟南芥At1g77740基因起始密码子前487 bp处,该基因编码磷脂酰肌醇-4-磷酸 -5-激酶(AtPIP5K2),共分离分析表明T-DNA插入与盐不敏感性紧密连锁.以野生型拟南芥总RNA为模板,克隆拟南芥AtPIP5K2基因cDNA,其开放读码框为2 265bp,编码755个氨基酸.与已报道物种PIPKs基因氨基酸序列比较分析表明,AtPIP5K2与植物PIPKs基因氨基酸相似性高达62%～75%,但与其他生物物种PIPKs基因之间的氨基酸相似性仅为33%～37%;AtPIP5K2推导的氨基酸序列中含有植物PIPKs基因所具有的高度保守区域“PIPKc"、“MORN repeat".进一步分析表明AtPIP5K2基因在拟南芥根及莲座叶片中表达量较强,并且由于T-DNA的插入,使eto突变株系与野生型相比,其AtPIP5K2基因过量表达,表明AtPIP5K2基因编码的产物可能参与调节拟南芥适应盐胁迫的调节反应.      

一个新的水稻TE的克隆及其与CACTA转座子结构的比较

对水稻受稻瘟病菌诱导的新因子Rim2的基因组结构进行了研究.以Rim2 cDNA片段作探针, 筛选水稻BAC文库并对其亚克隆, 获得了一个基因组DNA克隆Rim2-569.序列分析表明, Rim2-569序列具备Class 2 转座子的基本结构特征.它两端具有完整的末端颠倒重复(TIRs), 若干正向和反向的亚末端重复 (STRs) , 以及插入位点3 bp的同向重复.它TIRs上的保守序列CACTG有别于以往报道的CACTA转座子.该因子包含一个开读框, 其预测蛋白与CACTA转座子编码的TNP2、TNPD等转座酶有低程度的同源性.该因子没有能够编码类似TNP1/TNPA 的DNA结合蛋白的开读框.Southern杂交显示, Rim2因子在多个不同起源的水稻品种上广泛存在,连同检索结果,证明该家族具有很多拷贝.上述特点表明, 该因子属于一个与CACTA转座子有一定差异的新的转座子大家族.     

Probing the component structure of a maize gene with transposable elements

Three instances of R gene instability were found in maize stocks carrying the controlling elements Dissociation (Ds) and Modulator (Mp). In each, Ds or a Ds-like element had transposed to R, inhibiting kernel pigmentation irregularly. When Mp was removed from the genome, R expression stabilized at lowt to intermediate levels. Strong pigmenting action was restored through recombination in heterozygotes of the three new forms with an R allele that specifies only plant pigmentation. The sites of Ds insertion mapped distal to the region that specifies seed versus plant expression. The evidence suggests that an R functional unit consists of one component that both governs tissue-specific expression and another that is common to alleles of different tissue-specific activities.     

基于高通量测序组装‘赤霞珠’叶绿体基因组及其特征分析

【目的】以欧亚种葡萄‘赤霞珠’(Cabernet Sauvignon)为试材,建立适于葡萄属(Vitis)植物完整叶绿体基因组组装及其特征分析的方法,为研究葡萄属植物的进化和系统发育提供方法指导。【方法】采用Illumina Hi Seq PE150双末端测序策略对其全基因组DNA建库测序,建库类型为350 bp DNA小片段文库,测序深度为10倍。以已发表的拟南芥(Arabidopsis thaliana)和欧亚种葡萄‘黑比诺’(Pinot Noir)的叶绿体基因组序列为参考,通过BLASTN比对提取葡萄叶绿体基因组序列,并用SOAPdenovo软件进行组装,得到‘赤霞珠’完整的叶绿体基因组并对其进行特征分析。【结果】基于高通量Illumina测序,共获得5.2 G的全基因组原始数据,其中,葡萄叶绿体基因组序列为0.42 G,约占全基因组序列的8%。用抽提出来的葡萄叶绿体基因组序列成功组装出‘赤霞珠’完整叶绿体基因组。特征分析表明,叶绿体基因组序列全长160 676 bp,包括大单拷贝区(large single copy,LSC)、小单拷贝区(small single copy,SSC)和2个反向重复序列(inverted repeat,IRA和IRB),长度分别为89 134、19 072和26 235 bp,具有典型被子植物叶绿体基因组环状四分体结构;共注释得到154个基因,包括99个蛋白编码基因、47个t RNA基因和8个r RNA基因;其叶绿体基因组的GC含量为37.43%;共检测到37个串联重复序列(tandem repeat sequence)和53个散在重复序列(dispersed repeats),其中,绝大部分串联重复序列的长度为11—42 bp,占叶绿体基因组序列的0.83%,而散在重复序列占叶绿体基因组序列的5.33%;此外,还检测到50个简单重复序列(simple sequence repeats,SSR)位点,大部分的SSRs均由A或T组成,同时SSRs在‘赤霞珠’叶绿体基因组上的分布是不均匀的,LSC区段含有39个SSRs,而SSC区段和IR区段分别仅有7个和4个SSRs;与蛋白编码基因对应的密码子偏好使用A/T碱基,并且编码亮氨酸(L)的密码子使用频率最高,而编码半胱氨酸(C)的密码子使用频率最低;系统发育分析表明‘赤霞珠’与‘黑比诺’、夏葡萄(Vitis aestivalis)、圆叶葡萄(Vitis rotundifolia)亲缘关系最近。【结论】基于全基因组高通量测序的方法,成功组装出‘赤霞珠’完整的叶绿体基因组,与传统获得叶绿体基因组的方法相比,此方法不需要分离叶绿体和提取cpDNA,缩短了试验时间、降低了劳动强度,并且极大地提高了试验的可行性。‘赤霞珠’叶绿体基因组的基因结构、基因顺序、GC含量和密码子偏好性均与典型的被子植物叶绿体基因组类似。     

蒺藜苜蓿叶绿体微卫星分布规律的研究

利用已公布的蒺藜苜蓿(Medicao truntula)叶绿体DNA(cpDNA)全序列测序结果,应用生物信息学软件对蒺藜苜蓿微卫星位点的分布情况进行了统计分析,结果表明:在已公布的124 033 bp的蒺藜苜蓿叶绿体基因组序列中,共有341个SSR序列,SSR的碱基总数达3 987 bp,约占整个基因组的3.2%;在所有SSR序列中,数量最多的是单碱基SSR,数量达到297个,占总数的87.1%,其次是二碱基重复序列,占12.6%,三碱基微卫星序列最少,仅有1个,大于三碱基的微卫星数为0;在单碱基重复中又以A和T重复为主,占单碱基重复总数的84.5%,二碱基重复也以AT和TA为主,占95.5%,单碱基中的纯粹重复类型占总数的65.3%。研究结果为该物种的分子标记的筛选提供了基础信息。     

稻曲病菌T-DNA插入突变菌株B1241侧翼基因克隆

【目的】分析稻曲病菌(Ustilaginoidea virens)致病力减弱的T-DNA插入突变菌株B1241的生物学性状和致病力,并结合分子生物学手段研究其T-DNA插入位点的侧翼基因,以解析突变基因在稻曲病菌生长和致病过程中的作用,从而为阐明稻曲病菌致病机制提供理论基础。【方法】以野生菌株P1作为对照,观察并检测突变菌株B1241的菌落形态、生长速率、孢子形态、产孢量等生物学性状;采用注射接种的方法将菌丝和孢子的混合液接种于水稻穗苞中,统计每穗发病的病粒数,分析B1241的致病性变化;突变菌株B1241在不含有潮霉素的PSA平板上转接5代之后,PCR检测T-DNA插入的稳定性并通过Southern杂交分析B1241中T-DNA插入的拷贝数;利用HiTail-PCR获得T-DNA插入位点的侧翼序列,经NCBI比对得到侧翼基因;运用RACE-PCR克隆插入位点侧翼基因全长;qRT-PCR分析侧翼基因的表达情况。【结果】经生物学特性观察及田间接种发现,与野生菌株P1相比,突变菌株B1241在固体培养基MM、PSA和TB3上的菌落和孢子形态以及生长速率无显著差异,其致病力、产孢能力均呈极显著下降。B1241在不含潮霉素的PSA平板上转接5代之后,仍能扩增到GFP和HPH基因,说明T-DNA已经稳定地插入到其基因组中。Southern杂交结果显示T-DNA在该突变菌株中以单拷贝的形式插入。经扩增并比对侧翼序列,T-DNA的插入位点处少了28 bp的稻曲序列,有37 bp在T-DNA及稻曲病菌基因组中都没有比对到。NCBI比对发现,侧翼基因Uvt-1241与UV-8b菌株的UV8b-7878基因同源,开放阅读框长2 317 bp,包含81和106 bp的2个内含子,编码709个氨基酸。经RACE-PCR获得基因全长2 650 bp,5'非编码区长度为14 bp,3'非编码区长度为319 bp。T-DNA插入在基因Uvt-1241的启动子区域,位于起始密码子之前516 bp处。qRT-PCR分析结果表明,Uvt-1241在该突变体中表达量下降。该基因编码一个糖基水解酶18家族的蛋白,同时含有一个保守结构域D××D×D×E。【结论】稻曲病菌突变菌株B1241中,T-DNA插入到基因Uvt-1241的启动子区域,从而导致该启动子功能部分缺失,基因表达量下降,使得突变菌株的生长、产孢等生物学特性及致病力发生改变,由此推测该基因可能在稻曲病菌生长及致病过程中起着重要的作用。     

甘蓝型油菜丙酮酸激酶基因RNA干扰载体的构建

采用基因芯片技术,从油菜中鉴定了一个油分积累优势表达基因即丙酮酸激酶（Pyruvate kinase, PK）基因。为构建甘蓝型油菜PK基因的RNA干扰（RNAi）载体,通过设计引物克隆了 PK基因长498 bp的 siRNA靶序列,连接到 pEASY-T1载体上,采用 NotI和XhoI酶切,酶切产物连接到本课题组新近改造的 RNAi 平台载体 pHurricane 的 NotI 和XhoI位点,通过多重 PCR 鉴定证实载体构建成功,然后将上述 PK基因反向重复框克隆到加入 napin启动子的 pCAMBIA1390表达载体相应的酶切位点上,构建具有种子特异性表达的PK基因的RNAi载体。限制性内切酶酶切证实载体构建成功,为进一步研究 PK基因参与决定油菜油分积累的分子机理和代谢调控奠定了基础。     

水稻分离群体中半矮秆和致死性黄苗突变体的形成机理研究

[目的]鉴定sdwrky和lysrcc1突变体的Ds插入突变基因,初步分析sdwrky和lysrcc1突变体形成的分子机理。[方法]采用TAIL-PCR技术,分别从sdwrky突变体和lysrcc1突变体克隆Ds侧翼基因序列,并进行序列分析。[结果]Ds分别插入sdwrky突变体4号染色体Os04g0597300（sdwrky）基因和lysrcc1突变体3号染色体Os03g0599600（lysrcc1）基因,导致sdwrky和lysrcc1基因突变。[结论]sdwrky基因编码包含WRKY结构域的蛋白质（SDWRKY）,推测SDWRKY与OsWRKY24具相似功能,作为糊粉层细胞内ABA和GA信号传导途径中共同的抑制因子,调控水稻种子萌发及幼苗生长,Sdwrky基因突变导致形成sdwrky突变体。lysrcc1基因编码包含RCC1结构域的蛋白质（LYSRCC1）,LYSRCC1可能作为RanGEF参与核质运输,影响叶绿体合成的某一环节,lysrcc1基因隐性突变导致形成ylrcc1突变体。