高粱高效Ubiquitin启动子的克隆及活性鉴定

在转基因植物的研究中，启动子是影响转基因表达效率的重要因素之一。植物泛素（ubiquitin）基因启动子以启动效率高、甲基化程度相对较低、遗传性状稳定等特点成为单子叶植物中应用较为广泛的启动子。其中，玉米的Ubi-1是最常使用的Ubiquitin启动子之一。本研究旨在克隆高粱高效Ubiquitin启动子，为高粱及其他单子叶植物的遗传转化研究提供新的组成型启动子选择。本研究从NCBI数据库中查找到多个polyubiquitin基因，下载其起始密码子上游3000 bp的序列。根据Ubiquitin启动子的特点，选择含有5′ UTR内含子且大小合适的序列，并通过在线启动子预测网站进行启动子分析，最后选择2个基因（LOC8076096、LOC8063786）进行启动子克隆，将其启动子序列分别命名为U1和U5。将这2个启动子片段PCR扩增后，连入含有GUS报告基因的植物表达载体Ubi-GUS，得到重组表达载体U1-GUS和U5-GUS。重组载体通过农杆菌介导法转化水稻和狗尾草，PCR筛选阳性转化苗，对阳性转化苗进行GUS染色分析，鉴定U1和U5的启动子活性。GUS染色观察发现，所有以U1和U5为启动子的水稻和狗尾草阳性转化苗的根、茎和叶均呈现较深的蓝色，比以玉米Ubi-1为启动子的阳性转化苗的蓝色略深，且以U5为启动子的阳性转化苗比以U1为启动子的蓝色更深，而非转基因的水稻和狗尾草小苗则完全染不上蓝色。因此，启动子U1和U5在水稻和狗尾草中均具有组成型强启动子的活性，可作为新的组成型启动子应用于高粱、水稻、狗尾草及其他单子叶植物的遗传转化研究。     

水稻种子特异性谷蛋白GluB1启动子在水稻愈伤组织中驱动外源基因表达

【目的】水稻谷蛋白启动子GluB1(GluB1promoter,pGluB1)常用于外源基因在种子中特异性高效表达的研究,也是研究种子储藏蛋白基因表达调控机制的模型。前人研究表明,pGluB1只在水稻胚乳中表达,而在根、茎、叶片、叶鞘、颖壳等组织中均无表达活性。研究的目的是为了克服种子特异表达启动子筛选周期长的缺点。【方法】将由pGluB1驱动的霍乱毒素B亚单位和重组胰岛素原组成的融合基因(a fusion gene of the cholera toxin B subunit and human proinsulin, CTBIN)表达载体pCAMBIA1302-pGluB1sig-CTBIN-NOS经农杆菌介导法转化水稻成熟胚愈伤组织。通过RT-PCR和蛋白质印迹杂交试验检测融合基因CTBIN在水稻愈伤组织中的转录和翻译表达。【结果】获得的7个转基因愈伤克隆中,有6个克隆的融合基因CTBIN在转录水平上表达。选取其中4个克隆进一步进行蛋白质印迹杂交试验检测证实融合基因CTBIN均在翻译水平上表达,而且从分子量大小推断融合蛋白包含的谷蛋白GluB1的N-端信号肽序列(24个氨基酸残基)在所测的愈伤组织细胞中均被成功切除。【结论】水稻种子特异性启动子pGluB1在愈伤组织中具有驱动外源基因表达活性,种子蛋白体亚细胞定位信号肽序列可在愈伤组织细胞中被切除。这为在愈伤组织细胞中快速检测种子特异表达启动子活性和探索愈伤组织中蛋白质的亚细胞分拣机制奠定了基础。     

花生中三个LEA基因的克隆与表达分析

胚胎发生晚期丰富蛋白(LEA)是一类受发育阶段及脱水信号调节的脱水保护蛋白,在响应植物干旱、低温、高盐等逆境胁迫中具有重要功能。本研究在转录组数据分析时,发现3个LEA基因在花生干旱胁迫时大量上调表达。利用RACE技术以及生物信息学方法,发现这三个基因分别属于LEA2、LEA3以及LEA4。通过序列比对进行进化分析,结果表明其均与Arachis duranensis,A.ipaensis相关基因具有极高相似度,印证了栽培花生来源于Arachis duranensis和A.ipaensis的研究结果。通过表达分析发现,正常状态下这三个基因在花生根、茎、叶中均有表达;干旱胁迫状态下,花生根部的LEA基因大量表达,暗示其在花生抗旱机制中发挥重要作用。本研究结果可为筛选新的抗旱花生种质提供理论依据。     

Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice.     

Promoter Trapping in Microalgae Using the Antibiotic Paromomycin as Selective Agent

The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.     

Isolation and characterisation of a xylanase inhibitor Xip-II gene from durum wheat

Cereals contain xylanase inhibitor proteins (XIPs) which inhibit microbial xylanases from glycoside hydrolase families 10 and 11. Here, we report for the first time the isolation and characterisation of a genomic clone containing a xylanase inhibitor gene. This gene, Xip-II, isolated from a durum wheat genomic library (Triticum durum Desf.) encodes a mature protein of 307 amino acid (aa) residues that shares highest aa sequence identity (64%) with the rice RIXI xylanase inhibitor. XIP-II showed inhibition against family 11 xylanases and no chitinase activity. In silico analysis of the 5′ promoter region of Xip-II revealed sequences with similarity to known cis regulatory elements upstream from the initiation codon. In particular, the identification of a number of cis-acting elements controlling the expression of defence and seed-specific genes supports the role for this class of inhibitors in plant defence against pathogens but also provides new clues on a potential role in plant development.     

小麦LEAsm基因及其启动子的克隆与功能研究

胚胎发育晚期丰富蛋白(LEA)是一个小的高亲水性的蛋白家族,该蛋白家族在逆境胁迫下大量积累,保护植物免受逆境胁迫。LEA蛋白可分为7组,其中重复的11-氨基酸基序是第3组LEA蛋白的特征。为深入分析第3组LEA蛋白在小麦响应逆境胁迫中的作用机制,利用芯片技术从小麦表达谱中筛选出一个渗透胁迫诱导表达的第3组LEA蛋白基因TaLEAsm,然后根据该基因序列设计引物筛选石麦15的BAC文库,获得1个含有该基因的BAC单克隆,以该BAC单克隆质粒为模板,通过BAC延伸测序克隆了TaLEAsm基因及其启动子序列,并对TaLEAsm序列特征、表达模式和启动子功能进行了初步分析。结果表明,TaLEAsm基因序列仅含有1个105bp的内含子,其开放读码框长675bp,编码224个氨基酸。TaLEAsm含有10个11-氨基酸重复序列,属于第3组LEA蛋白。低温、高盐和渗透胁迫均诱导TaLEAsm基因上调表达,但在根和叶中表达模式不同。在TaLEAsm基因起始密码子上游1 500bp序列中,预测含有14个逆境响应顺式元件。在拟南芥中,TaLEAsm基因启动子能够启动GUS基因表达,渗透胁迫诱导GUS基因明显上调表达。以上结果表明,TaLEAsm为小麦脱水响应基因,其启动子为渗透胁迫诱导启动子。     

Genome-Wide Analysis of von Willebrand Factor A Gene Family in Rice for Its Role in Imparting Biotic Stress Resistance with Emphasis on Rice Blast Disease

von Willebrand factor A (vWA) genes are well characterized in humans except for few BONZAI genes, but the vWA genes are least explored in plants. Considering the novelty and vital role of vWA genes, this study aimed at characterization of vWA superfamily in rice. Rice genome was found to have 40 vWA genes distributed across all the 12 chromosomes, and 20 of the 40 vWA genes were unique while the remaining shared large fragment similarities with each other, indicating gene duplication. In addition to vWA domain, vWA proteins possess other different motifs or domains, such as ubiquitin interacting motif in protein degradation pathway, and RING finger in protein-protein interaction. Expression analysis of vWA genes in available expression data suggested that they probably function in biotic and abiotic stress responses including hormonal response and signaling. The frequency of transposon elements in the entire 3K rice germplasm was negligible except for 9 vWA genes, indicating the importance of these genes in rice. Structural and functional diversities showed that the vWA genes in a blast-resistant rice variety Tetep had huge variations compared to blast-susceptible rice varieties HP2216 and Nipponbare. qRT-PCR analysis of vWA genes in Magnaporthe oryzae infected rice tissues indicated OsvWA9, OsvWA36, OsvWA37 and OsvWA18 as the optimal candidate genes for disease resistance. This is the first attempt to characterize vWA gene family in plant species.     

乔松根特异启动子PmPgPR10驱动Na+/H+逆转运蛋白提高转基因水稻的耐盐性

 为获得抗盐水稻,将小麦液泡膜Na+/H+逆向转运蛋白基因TaNHX2与乔松(Pinus griffithii)根诱导型特异表达启动子PmPgPR10融合（PmPgPR10∷TaNHX2）并转化水稻,以研究PmPgPR10启动子对TaNHX2基因表达的影响以及转基因植物的耐盐性。PCR、Southern和实时PCR试验结果表明,PmPgPR10∷TaNHX2基因已通过农杆菌介导法整合进水稻基因组,而且外源基因已在受体细胞中正确表达。在盐胁迫处理时,转PmPgPR10基因植株的耐盐性以及外源基因的表达量显著高于对照植株,说明PmPgPR10启动子可以调控TaNHX2基因在根中特异表达。为了进一步分析转基因植株耐盐机理, 比较了日本晴和转基因T3代植株中液泡腺苷三磷酸酶（V ATPase）和液泡焦磷酸酶（V PPase）活性,发现转PmPgPR10∷TaNHX2基因水稻的V ATPase和V PPase酶活性显著高于非转基因对照,说明V ATPase和V PPase活性提高在转TaNHX2基因水稻耐盐性过程中发挥重要作用。在盐胁迫处理时,V ATPase和V PPase活性只能在转基因植株的根中但不能在叶片中被检测到,进一步说明PmPgPR10启动子在根中特异性表达。因此,PmPgPR10具有在根中增强下游TaNHX2基因表达,并显著提高转基因植株耐盐性的能力。     

高等植物WRKY转录因子家族的演化及功能研究进展

WRKY转录因子是植物转录调节因子的最大家族之一,并且是调节植物许多生物过程的信号网络的组成部分。WRKY转录因子通过其保守结构域与靶基因启动子区域的W-box特异性结合,调节靶基因的表达,进而调控植物的叶片衰老、种子萌发与休眠、开花等生长发育过程外,还参与调控植物生物和非生物胁迫的响应过程。本文用代表性植物基因组数据,对WRKY的基因演化作了归纳,综述了近二十年来国内外WRKY转录因子的相要研究进展,并介绍了该转录因子在植物生物胁迫和非生物胁迫应答及生长发育过程中的调控作用。      

Interference Efficiency and Effects of Bacterium-mediated RNAi in the Fall Armyworm (Lepidoptera: Noctuidae)

RNAi is an effective tool for gene function analysis and a promising strategy to provide environmentally friendly control approaches for pathogens and pests. Recent studies support the utility of bacterium-mediated RNAi as a cost-effective method for gene function study and a suitable externally applied delivery mechanism for pest control. Here, we developed a bacterium-mediated RNAi system in Spodoptera frugiperda based on four target genes, specifically, Chitinase (Sf-CHI), Chitin synthase B (Sf-CHSB), Sugar transporter SWEET1 (Sf-ST), and Hemolin (Sf-HEM). RNAi conducted by feeding larvae with bacteria expressing dsRNAs of target genes or injecting pupae and adults with bacterially synthesized dsRNA induced silencing of target genes and resulted in significant negative effects on growth and survival of S. frugiperda. However, RNAi efficiency and effects were variable among different target genes and dsRNA delivery methods. Injection of pupae with dsCHI and dsCHSB induced a significant increase in wing malformation in adults, suggesting that precise regulation of chitin digestion and synthesis is crucial during wing formation. Injection of female moths with dsHEM resulted in lower mating, fecundity, and egg hatching, signifying a critical role of Sf-HEM in the process of egg production and/or embryo development. Our collective results demonstrate that bacterium-mediated RNAi presents an alternative technique for gene function study in S. frugiperda and a potentially effective strategy for control of this pest, and that Sf-CHI, Sf-CHSB, Sf-ST, and Sf-HEM encoding genes can be potent targets.     

Rice Germin-Like Proteins: Allelic Diversity and Relationships to Early Stress Responses

Germin-like protein (GLP) markers were associated with quantitative trait loci (QTL) for resistance to the rice blast pathogen, Magnaporthe oryzae in multiple rice (Oryza sativa) mapping populations. Twelve paralogous OsGLP gene family members are located within the physical QTL region on chromosome 8, and gene silencing studies suggest that they contribute collectively to the resistance phenotype. We compared sequence and expression profiles of OsGLP alleles in two resistant and two susceptible parental rice lines to find functional polymorphisms that correlated with the resistant phenotype. Based on coding and promoter sequences, the genes belong to two germin subfamily groups (GER3 and GER4). OsGLP members from both subfamilies were constitutively expressed and developmentally regulated in all cultivars. Transient induction above constitutive levels was observed for some OsGLPs, especially GER4 subfamily members, at early time points after M. oryzae infection and mechanical wounding. Varying 5′ regulatory regions and differential expression of some family members between resistant and susceptible cultivars corresponded with differential hydrogen peroxide (H₂O₂) accumulation after the same stimuli. OsGLP of both GER subfamilies localized to the plant cell wall. The protein location and early gene induction suggest that OsGLPs protect rice leaves at early stages of infection before fungal penetration and subsequent ingress. Our data suggest that regulation of OsGLP genes defines resistant versus susceptible phenotypes.     

基于Web界面的水稻基因芯片注释整合数据库

开发了1个基于Web界面在生物学背景下注释水稻基因芯片的整合数据库RiceDB,它的注释信息比同类针对水稻Affymetrix 57K全基因组表达谱芯片的注释数据库更全面、更快捷且可以自动更新。它由8个功能模块组成,RiceMap建立水稻Affymetrix 57K探针组与基因的对应关系,从而提供来自各个水稻生物信息学数据库功能描述信息;RiceGO整合Gene Ontology数据库从生物学途径/分子功能和细胞构建对Affymetrix 57K探针组进行注释;RiceKO确定Affymetrix 57K探针组代表的基因可能参与的代谢与调控路径;RiceUP、RiceDO、RiceMR、RiceCD、RiceGF分别确定Affymetrix 57K探针组代表的基因上游小于1 kb启动子序列、结构功能域组成、受哪些水稻MicroRNA调控、染色体定位、基因家族分类。     

International Consortium of Rice Mutagenesis: resources and beyond

Rice is one of the most important crops in the world. The rice community needs to cooperate and share efforts and resources so that we can understand the functions of rice genes, especially those with a role in important agronomical traits, for application in agricultural production. Mutation is a major source of genetic variation that can be used for studying gene function. We will present here the status of mutant collections affected in a random manner by physical/chemical and insertion mutageneses.As of early September 2013, a total of 447, 919 flanking sequence tags from rice mutant libraries with T-DNA, Ac/Ds, En/Spm, Tos17, nDART/aDART insertions have been collected and publicly available. From these, 336,262 sequences are precisely positioned on the japonica rice chromosomes, and 67.5% are in gene interval. We discuss the genome coverage and preference of the insertion, issues limiting the exchange and use of the current collections, as well as new and improved resources. We propose a call to renew all mutant populations as soon as possible. We also suggest that a common web portal should be established for ordering seeds.     

Prediction and Expression Analysis of miRNAs Associated with Heat Stress in Oryza sativa简

Computational prediction of potential microRNAs (miRNAs) and their target genes was performed to identify the miRNAs and genes associated with temperature response in rice. The data of temperature-responsive miRNAs of Arabidopsis, and miRNAs and the whole genome data of rice were used to predict potential miRNAs in Oryza sativa involved in temperature response. A total of 55 miRNAs were common in both the species, and 27 miRNAs were predicted at the first time in rice. Target genes were searched for these 27 miRNAs in rice genome following stringent criteria. Real time PCR based on expression analysis of nine miRNAs showed that majority of the miRNAs were down regulated under heat stress for rice cultivar Nagina 22. Furthermore, miR169, miR1884 and miR160 showed differential expression in root and shoot tissues of rice. Identification and expression studies of miRNAs during heat stress will advance the understanding of gene regulation under stress in rice.     

Phylogenomic Relationships of Rice Oxalate Oxidases to the Cupin Superfamily and Their Association with Disease Resistance QTL

Rice oxalate oxidase genes (OXO) may play a role in resistance to Magnaporthe oryzae. Genome analyses showed four tandemly duplicated OXO genes, OsOXO1–OsOXO4, which mapped to a blast resistance QTL in chromosome 3. These genes have >90% nucleotide and amino acid identity, but they have unique gene structures, conserved motifs, and phylogeny compared to the 70 other members of the cupin superfamily in the Nipponbare genome, which were divided into several classes. In resistant and susceptible Vandana/Moroberekan advanced backcross lines, only OsOXO4 was expressed during rice–M. oryzae interactions, and its expression increased earlier in resistant than susceptible lines. The earlier expression of OsOXO4 in resistant lines correlated with a 26-bp promoter insertion containing an additional copy of the bacterial responsive nodulation cis-element. Our results showed that OsOXO1–4 are in a separate class of rice cupin genes and supports a role for the promoter variant of OsOXO4 in resistance to M. oryzae.     

CRISPR/Cas9-Targeted Knockout of Rice Susceptibility Genes OsDjA2 and OsERF104 Reveals Alternative Sources of Resistance to Pyricularia oryzae

Rice genes OsDjA2 and OsERF104, encoding a chaperone protein and an APETELA2/ ethylene-responsive factor, respectively, are strongly induced in a compatible interaction with blast fungus, and also have function in plant susceptibility validated through gene silencing. Here, we reported the CRISPR/Cas9 knockout of OsDjA2 and OsERF104 genes resulting in considerable improvement of blast resistance. A total of 15 OsDjA2 (62.5%) and 17 OsERF104 (70.8%) T₀ transformed lines were identified from 24 regenerated plants for each target and used in downstream experiments. Phenotyping of homozygous T₁ mutant lines revealed not only a significant decrease in the number of blast lesions but also a reduction in the percentage of diseased leaf area, compared with the infected control plants. Our results supported CRISPR/Cas9-mediated target mutation in rice susceptibility genes as a potential and alternative breeding strategy for building resistance to blast disease.