油菜BnCr4基因RNAi载体构建及植株转化

该实验构建了含甘蓝型油菜黄化相关基因BnCr4特异片段反向重复结构的RNA干扰(RNAi)载体pFGC5941-Cr4,通过根癌农杆菌介导转化油菜,获得47株抗Basta的抗性再生油菜植株,其中10株经PCR鉴定为阳性转基因植株.随机选取3株经鉴定的转基因阳性油菜植株进行半定量RT-PCR分析,结果显示,相对于非转基因的野生型油菜,3株转基因植株中BnCr4基因的表达量分别降低了78.5%、8.5%、11.8%,表明该干扰载体转入油菜能特异引起植株BnCr4基因表达量下降.     

甘蓝型油菜MADS-box基因家族的鉴定与系统进化分析

MADS-box基因家族参与调控开花时间、花器官分化、根系生长、分生组织分化、子房和配子发育、果实膨大及衰老等植物生长发育的重要过程。基于甘蓝型油菜(Brassica napus)基因组测序数据,利用生物信息学方法对甘蓝型油菜MADS-box基因家族进行鉴定和注释及基因结构与系统进化分析。结果显示,在甘蓝型油菜中鉴定出307个MADS-box基因家族成员,根据进化关系可将其分为两大类型,I型(M-type)包含α、β、γ三个亚家族,II型(MIKC-type)包括MIKCC和MIKC*两个亚家族,MIKCC可进一步分为13个小类;甘蓝型油菜A基因组染色体上分布的MADS-box基因多于C基因组。在基因结构上,MIKC-type亚家族基因序列普遍比M-type长且含有较多的外显子;M-type亚家族蛋白序列中的motif数量为2–5个,MIKC-type亚家族蛋白序列中平均含有7个motif。拟南芥(Arabidopsis thaliana)与甘蓝型油菜MADS-box基因共线性分析结果显示,全基因组复制事件对MADS-box基因家族尤其是MIKC亚家族的扩张起重要作用;MIKC亚家族基因在进化过程中受到的选择压力约为M-type的2倍,这表明MIKC-type亚家族在进化过程中被选择性保留。     

甘蓝型油菜MAP70基因家族全基因组鉴定与表达分析

采用生物信息学方法,从甘蓝型油菜（Brassica napus L.）基因组数据库中筛选鉴定MAP70s基因家族成员,对鉴定得到的BnaMAP70s基因家族成员的序列特征进行生物信息学分析,同时利用qRT-PCR方法分析该基因家族成员在不耐渍和强耐渍甘蓝型油菜品种幼苗中的基因表达水平。结果显示,本研究共鉴定得到19个BnaMAP70基因家族成员,分布在11条染色体上,可分为5个亚家族。BnaMAP70s启动子的上游存在厌氧胁迫、响应植物激素等相关元件,表明BnaMAP70s可能参与植株生长发育和渍水胁迫调控;转录水平存在品种特异性、植株部位特异性、时间特异性。q RT-PCR分析结果表明,BnaMAP70-1和BnaMAP70-4在渍水胁迫下出现表达差异,说明这两个基因受渍水胁迫的调控。     

油菜Bna miR1140基因的分离与转化及其过表达分析

为了探究油菜miR1140基因的功能,该研究利用PCR技术从油菜栽培种westar中克隆了miR1140前体基因,并与pPZP212载体连接,构建了miR1140前体基因的过表达载体,通过农杆菌介导法将其转化到甘蓝型油菜westar品种中。(1)PCR鉴定表明,共获得14株过表达Bna-miR1140基因油菜阳性株。(2)表型观察显示,阳性株的叶片形态、花器结构没有变异,株高、主花序长度、主花序结角数和千粒重均与对照相当,但其中有5株过表达Bna-miR1140基因油菜阳性株(T0代)出现双主序株型,且分枝部位较对照低,分枝数较对照明显增多,使全株有效角果数增多,单株生产力较对照提高26%,而其他9株阳性株均与野生型油菜表型一致。(3)T1代田间表型分析表明,5株过表达35S∷Bna-miR1140(T0代)的T1代转化株系全生育期较对照极显著延长9~14d,且5株T0代中有4株的株型变异符合3∶1的遗传分离规律。研究推测,油菜Bna-miR1140基因可能参与了调控油菜的分枝发育。     

利用农杆菌介导法将γ-生育酚甲基转移酶基因导入油菜的研究

将γ-生育酚甲基转移酶基因导入油菜,旨在提高菜油中的维生素E含量。为了使该基因在油菜种子中特异表达,采用油菜种子特异启动子BcNA1,构建了植物表达载体pBIBE。以花油五号和花油六号油菜品种子叶柄为受体,将γ-生育酚甲基转移酶基因(γ-TMT)通过农杆菌介导法转化油菜,获得了22株抗卡那霉素再生植株。经过PCR检测,其中有7株表现为阳性,初步证明γ-TMT已整合到油菜基因组中。     

茄科植物木糖转移酶家族基因挖掘与进化分析

木糖转移酶在植物多糖合成以及蛋白糖链修饰中起重要作用。本研究试图从全基因组水平上系统鉴定植物木糖转移酶基因保守结构域,并挖掘茄科植物中木糖转移酶家族基因新成员。研究中选取了5种茄科植物,分析鉴定了茄科植物中的木糖转移酶基因及其保守结构域,并构建得到了系统发育树。系统鉴定获得了54条木糖转移酶家族基因。其中,矮牵牛基因组中含有6条家族成员,而在本氏烟中含有11条成员。本家族成员可分为5个亚家族,其中的两个亚家族各自能进一步分为两个显著的独立枝。在鉴定得到的基因中,仅有2条拟南芥基因功能得到了鉴定,分别参与蛋白糖基化和种皮多糖合成。拟南芥每条木糖转移酶基因仅含有1个保守结构域,而此结构域覆盖各基因大片区域。各基因编码蛋白分子量较大,且表现较强的疏水性。本研究为今后针对茄科植物木糖转移酶的深入研究提供了基因靶标。     

茎尖法转基因棉花植株真实性鉴定方法探究

棉花茎尖转化法具备不受基因型限制、转化周期短的优点,是理想的棉花转化体系,但据报道其所获得的转基因植株普遍存在遗传不稳定、高代植株基因丢失的现象。以陆地棉TM?1品种为受体材料,利用茎尖转化法将DsRed2载体转入棉花,经卡那霉素筛选获得16株抗性植株,进一步PCR扩增靶基因,获得6株DsRed2基因片段PCR检测为阳性的植株,初步判断该6株为茎尖转化法获得的转基因植株,但经紫外照射,6个转基因植株均未检测到红色荧光。对其进行靶基因RT?PCR,发现DsRed2基因在6个转基因植株中仅有极低量的表达或无表达。进一步对DsRed2载体的非T?DNA片段,即载体骨架部分进行PCR以及植株内生菌培养检测,结果表明,6个转基因植株均含有完整的DsRed2载体,植株可培养出含有完整载体的内生菌,且内生菌经农杆菌16S核糖体RNA（16S rRNA）片段PCR检测结果为阳性,推测由于茎尖侵染形成农杆菌与植株共生关系,造成假阳性株的现象,进而导致高代转基因植株基因丢失、遗传不稳定的现象。旨在建立一套完整的茎尖法转基因棉花植株真实性的鉴定方法,为进一步深入研究提供参考依据。     

铁皮石斛14-3-3基因家族鉴定及表达分析

【目的】14-3-3蛋白,亦称通用调节因子（GRF）,由多基因家族编码,在植物生长发育和逆境应答发挥关键的作用。鉴定铁皮石斛（Dendrobium officinale）GRF基因家族,为铁皮石斛GRF基因功能研究及遗传改良提供理论依据。【方法】通过生物信息学的方法鉴定铁皮石斛14-3-3家族成员,分析其理化性质、染色体定位、系统进化发育、基因结构和启动子顺式作用元件等,同时通过荧光定量PCR技术检测它们在不同组织、低温处理及盐胁迫处理后的表达量。【结果】铁皮石斛有17个GRF家族成员,分为ε类和非ε类亚族,不均匀地分布在7条染色体上,且存在7对串联复制基因。同一亚族成员基因结构、保守基序和蛋白质二级结构相类似。DoGRF家族基因的启动子区域存在大量激素和环境胁迫应答相关的调控元件。DoGRF家族基因在铁皮石斛各组织中均有表达,具有组织表达特异性,大多数基因在花器官中表达最高,其次是茎和根。同时,在低温处理、盐胁迫处理下呈现差异化表达,可能受到低温和盐胁迫的调控,特别是DoGRF2在铁皮石斛逆境应答过程中起着关键的作用。【结论】在全基因组水平从铁皮石斛中鉴定出17个DoGRF家族成员,...     

油菜EIN3/EIL基因家族的鉴定及结构分析

Ethylene-insensitive3(EIN3)和EIN3-like(EIL)蛋白是乙烯信号转导途径中重要的核转录因子。EIN3/EIL基因家族在高等植物中分布广泛,在植物的生长、发育等多个过程中有重要的作用。为了揭示油菜EIN3/EIL基因家族的生物学信息,利用生物信息学的方法鉴定了油菜EIN3/EIL基因家族的成员,并从理化性质、亚细胞定位、跨膜结构、二级结构等几个方面对油菜EIN3/EIL基因家族成员进行了预测和分析。这些研究结果有助于进一步研究油菜EIN3/EIL基因家族成员的功能,同时在生产实践中也具有一定的应用价值。     

枸杞Lb14-3-3c基因克隆及转化马铃薯的研究

在开花植物中,14-3-3蛋白对植物生长发育具有重要的调控作用。本试验利用逆转录PCR(RT-PCR)技术,从宁夏枸杞宁杞1号花药中克隆了一个14-3-3蛋白家族基因Lb14-3-3c。利用荧光定量PCR技术分析Lb14-3-3c基因在花药发育不同时期的表达特征,构建Lb14-3-3c基因的植物过表达载体pCambia1305.1-35s-Lb14-3-3c(+)及植物抑制表达载体pCambia1305.1-35s-Lb14-3-3c(-),继而经农杆菌介导法将过表达载体转化马铃薯紫花白幼茎,获得了阳性转基因马铃薯植株。结果表明,Lb14-3-3c基因编码的蛋白与番茄处于进化树同一分枝上,亲缘关系最近。荧光定量PCR分析表明,该基因在枸杞各器官都有表达,在雄蕊中的表达量最高。在花药发育的各个时期均表达,且在小孢子母细胞时期表达量最高。成功将外源基因Lb14-3-3c载入含强启动子CaMV35s的植物表达载体中,并将该基因的植物过表达载体转化马铃薯,通过表型观察与PCR阳性鉴定得到5株转基因植株,发现转基因植株长势优于野生型植株,苗期野生型与转基因型淀粉含量相差不大,结薯期和成熟期转基因型马铃薯的叶片淀粉含量均高于野生型,且差异显著。本研究为进一步探讨Lb14-3-3c基因对枸杞花药发育过程中淀粉供能的调控提供了参考依据,并且为阐明Lb14-3-3c基因在植物发育过程中的功能及枸杞的分子遗传改良提供了研究基础。     

Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37° C

We have analyzed the structure of genes encoding the glyoxylate cycle enzyme isocitrate lyase from Brassica napus L. and their expression during embryogeny and postgermination. Restriction mapping, nucleotide sequence, and DNA gel blot hybridization analyses of cDNA and genomic clones indicated that there are approximately six isocitrate lyase genes in the B. napus genome that can be divided into at least two subfamilies based upon their divergence in 5′ and 3′ untranslated regions. We showed previously that isocitrate lyase mRNA accumulates during late embryogeny and postgermination. Here, we present results which indicate that several isocitrate lyase genes are expressed at both stages of development. First, gene-specific probes were used to show that mRNAs encoded by representatives of both gene subfamilies accumulated in both late maturation stage embryos and in seedlings of B. napus. Second, a single B. napus isocitrate lyase gene, together with 3.5 kb and 1.4 kb of 5′ and 3′ flanking regions, respectively, was expressed in both embryos and seedlings of transgenic tobacco plants. The results indicated that accumulation of isocitrate lyase in late embryogeny and postgermination does not result from the alternate expression of distinct members of the gene family.     

Developmentally regulated site-specific marker gene excision in transgenic <Emphasis Type="Italic">B. napus</Emphasis> plants

We have developed a self-excision Cre-vector to remove marker genes from Brassica napus. In this vector cre recombinase gene and bar expression cassette were inserted between two lox sites in direct orientation. These lox-flanked sequences were placed between the seed-specific napin promoter and the gene of interest (vstI). Tissue-specific cre activation resulted in simultaneous excision of the recombinase and marker genes. The vector was introduced into B. napus by Agrobacterium-mediated transformation. F1 progeny of seven lines with single and multiple transgene insertions was subjected to segregation and molecular analysis. Marker-free plants could be detected and confirmed by PCR and Southern blot in all transgenic lines tested. The recombination efficiency expressed as a ratio of plants with complete gene excision to the total number of investigated plants varied from 13 to 81% dependent on the transgene copy number. Potential application of this system would be the establishment of marker-free transgenic plants in generatively propagated species.     

The Novel Use of a Combination of Sonication and Vacuum Infiltration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated Transformation of Kidney Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) with <Emphasis Type="Italic">lea</Emphasis> Gene

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.     

The EPSPS gene flow from glyphosate-resistant Brassica napus to untransgene B. napus and wild relative species Orychophragmus violaceus

Gene flow from transgenic plants to compatible wild relatives is one of the major impediments to the development of the culture of genetically engineered crop plants. In this work, the flow of EPSPS (conferring resistance to glyphosate) gene of transgene Brassica napus toward the untransgene B. napus and wild relative species Orychophragmus violaceus in an open field (1 ha) was studied. The data related to only the 2004 and 2005 autumn season on one location of southwest of China. Pollen dispersal and fertilization of the target plants were favored and a detailed analysis of the hybrid offspring was performed. In field, the data studied show that the gene flow frequency was 0.16% between GM and non-GM B. napus at a distance of 1 m from the transgenic donor area. The crosspollination frequency was 0.05% between GM and non-GM B. napus at a distance of 5 m from the transgenic donor area. At a distance of 10 m, no crosspollination was observed. According to the results of this study, B. napus transgene flow was low. However, the wild relative species O. violaceus could not be fertilized by the transgenic pollen of B. napus, no matter what the distance was.     

<Emphasis Type="Italic">MAM</Emphasis> gene silencing leads to the induction of C3 and reduction of C4 and C5 side-chain aliphatic glucosinolates in <Emphasis Type="Italic">Brassica napus</Emphasis>

Methylthioalkylmalate (MAM) synthases and their associated genes that have been extensively investigated in Arabidopsis control the side-chain elongation of methionine during the synthesis of aliphatic glucosinolates. A Brassica homolog of the Arabidopsis MAM genes was used in this study to analyze the role of MAM genes in B. napus through RNA interference (RNAi). The silencing of the MAM gene family in B. napus canola and B. napus rapeseed resulted in the reduction of aliphatic glucosinolates and total glucosinolate content. The results indicated that RNAi has potential for reducing glucosinolate content and improving meal quality in B. napus canola and rapeseed cultivars. Interestingly, MAM gene silencing in B. napus significantly induced the production of 2-propenyl glucosinolate, a 3-carbon side-chain glucosinolate commonly found in B. juncea mustard. Most transgenic plants displayed induction of 2-propenyl glucosinolate; however, the absolute content of this glucosinolate in transgenic B. napus canola was relatively low (less than 1.00 μmol g⁻¹ seed). In the high glucosinolate content progenies derived from the crosses of B. napus rapeseed and transgenic B. napus canola, MAM gene silencing strongly induced the production of 2-propenyl glucosinolate to high levels (up to 4.45 μmol g⁻¹ seed).     

Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development

In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family. This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development. The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported. One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangements in its 5 region and intron splicing sites. The remaining genes (Bp4A and Bp4C), as well as the cDNA clones, appear to code for small proteins of unique structure. Three different types of proteins can be predicted as a result of the deletion of carboxy or amino terminal portions of a conserved core protein. These proteins all share a common alternation of hydrophobic and hydrophilic domains. A fragment of the genomic clone containing the gene Bp4A, as well as the non-functional gene Bp4B, was introduced into tobacco plants via Agrobacterium-mediated transformation. The functional gene Bp4A is expressed in transgenic tobacco plants and shows spatial and temporal regulation consistent with the expression patterns seen in Brassica napus.     

Targeted modulation of sinapine biosynthesis pathway for seed quality improvement in Brassica napus

Arabidopsis thaliana and other members of the Brassicaceae accumulate the hydroxycinnamic acid esters sinapoylmalate in leaves and sinapoylcholine in seeds. Our recent understanding of the phenylpropanoid pathway although complex has enabled us to perturb the sinapine biosynthesis pathway in plants. Sinapine (sinapoylcholine) is the most abundant antinutritional phenolic compound in seeds of cruciferous species and therefore is a target for elimination in canola (Brassica napus) meal. We analysed A. thaliana mutants with specific blocks in the phenylpropanoid pathway and identified mutant lines with significantly altered sinapine content. Knowledge gained from A. thaliana was extended to B. napus and the corresponding phenylpropanoid pathway genes were manipulated to disrupt sinapine biosynthesis in B. napus. Based on our understanding of the A. thaliana genetics, we have successfully developed transgenic B. napus lines with ferulic acid 5-hydroxylase (FAH) and sinapoylglucose:choline sinapoyltransferase (SCT)-antisense. These lines with concomitant downregulation of FAH and SCT showed up to 90% reduction in sinapine. In addition to reduced sinapine content, we detected higher levels of free choline accumulation in the seeds. These results indicate that it is possible to develop plants with low sinapine and higher choline by manipulating specific steps in the biosynthetic pathway. These improvements are important to add value to canola meal for livestock feed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of a Novel Antiporter Gene from Brassica napus Resulted in Enhanced Salt Tolerance in Transgenic Tobacco Plants

Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na⁺/H⁺ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T₁ progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T₁ lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T₁ progenies could express _BnNHX1 and had salt tolerance at levels comparable to their T₀ parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.