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。     

利用外源钾通道基因改良水稻钾素营养

通过基因枪导入法,将外源钾通道基因 (KAT1和AKT1)导入水稻中花8号、中花9号、中花13以及8706中,获得了转基因植株,经分子检测,证实了钾通道基因在水稻基因组中的整合与表达。盆栽试验表明,转基因植株在低钾和高钾两种钾水平下其钾的累积能力都有明显提高。     

利用CRISPR/Cas9技术创制高效抗除草剂水稻

【目的】培育抗除草剂品种在水稻育种中具有重要意义。利用CRISPR/Cas9基因编辑技术,以黑龙江优质粳稻品种为材料,编辑乙酰乳酸合酶ALS基因,创制具有抗除草剂特性的水稻材料。【方法】利用CRISPR/Cas9技术,以乙酰乳酸合酶ALS为靶基因,构建单碱基突变载体pH-nCas9-PBE-ALS,以松粳22、龙粳46和绥粳18为转化材料,利用农杆菌介导转化获得转基因植株,通过对转基因植株的突变位点进行测序结合除草剂喷施试验,鉴定基因型及表型。【结果】经分子水平检测验证,获得ALS^S627N突变植株10株,ALS^S627N且¹⁸⁸⁴G-A但第628位氨基酸未改变突变植株1株,ALS^S627N/G628E突变植株1株。相较于野生型,以上三类突变植株均具有较强抗除草剂特性。【结论】利用CRISPR/Cas9基因编辑技术获得具有抗除草剂特性,能够稳定遗传,不含转基因标记的纯合株系,可为抗除草剂水稻育种提供基础材料。     

东乡野生稻苗期低温诱导表达新基因BGIOSGA013293-DX的过表达载体构建与转化

结合表达谱数据分析,参考公共数据库中的序列信息设计引物,利用RT-PCR技术,从东乡野生稻中获得了1个在低温诱导条件下高表达的螺旋—环—螺旋结构域蛋白基因BGIOSGA013293-DX的全长c DNA,并构建了其过表达载体。序列比对分析表明,该基因位点在93-11中编码308个氨基酸,而在东乡野生稻中编码335个氨基酸,比93-11多27个氨基酸。且这27个氨基酸连续分布在一起,另外还有1个氨基酸的差异。利用农杆菌介导法将BGIOSGA013293-DX转入水稻受体品种93-11,获得了9株过表达转基因水稻植株,经潮霉素抗性标记基因PCR阳性检测和GUS检测,获得的转基因植株为携带BGIOSGA013293-DX的阳性植株。     

过表达桃PpCuZnSOD基因提高大豆耐盐性研究

为研究桃(Prunus persica L.)自由基清除剂CuZnSOD编码基因对于大豆耐盐性的促进作用,将桃PpCuZnSOD全长基因克隆到连有35S启动子的表达载体上,并通过农杆菌导入大豆基因组中,通过PCR和Southern杂交方法检测阳性转基因植株,并对T2代转基因植株进行基因表达量分析和耐盐性评估。结果表明:转基因植株有较高的耐盐性,表现为种子萌发率和存活率高,叶绿素含量高。转基因植株在盐胁迫下存活时间更长,与非转基因植株相比,转基因植株的SOD、POD、CAT酶活性较高,而丙二醛含量显著降低。研究结果表明PpCuZnSOD基因过表达可以显著提高大豆植株的SOD活性水平,缓解盐胁迫对大豆的氧化损伤。     

玉米花粉管通道法转化脱水素BDN1基因

通过花粉管通道法将脱水素基因BDN1转入玉米自交系合344中,并对转化后代进行PCR和RT-PCR分子检测以及耐盐性功能鉴定,筛选耐盐性较高的转基因玉米新种质.结果表明,实验共获得88株T0代除草剂抗性植株,其中31株PCR检测呈阳性,14株RT-PCR检测呈阳性,对T4代转基因株系苗期进行300 mmol/L NaCl溶液的盐胁迫处理, 2个转基因株系耐盐性比对照提高两个级别.     

4个抗稻瘟病基因分子标记的建立及在水稻亲本中的分布

建立稻瘟病抗性基因的分子标记对于培育抗稻瘟病水稻品种有重要意义。本文利用抗稻瘟病基因Pi9、Pi2、Pi5和Pita基因序列与日本晴等位基因的序列差异,建立4个基因的共显性分子标记M-Pi9、M-Pi2、M-Pi5和M-Pita,并用这4个分子标记检测24个抗稻瘟病单基因系和48份水稻亲本,结合48份水稻亲本对广西采集的稻瘟病菌ZB1和ZB13苗瘟抗性鉴定结果,验证4个分子标记的特异性和有效性。结果表明,M-Pi9仅在含Pi9和Piz的单基因系中检测出特异带;M-Pi2仅在含Pi2和Pizt的单基因系中检测出特异带;M-Pi5仅在含Pi5、Pi3和Pii的单基因系中检测出特异条带;M-Pita在含Pita和Pita2的单基因系中检测出特异带。具有M-Pi2特异条带的水稻亲本对ZB1和ZB13均表现出抗性,而具有M-Pi9、M-Pi5或M-Pita特异条带的水稻亲本对ZB13均表现出抗性。     

盐胁迫对水稻的影响及水稻耐盐育种研究

盐胁迫是盐渍化地区水稻生产的主要制约因素之一。盐胁迫对水稻的形态发育及生理生化代谢产生不利影响,其作用机理主要表现在渗透胁迫和离子毒害等。耐盐水稻品种的选育方法主要是筛选耐盐材料,将常规选育和转基因、分子标记辅助选择等现代生物技术相结合,选育耐盐性强且具有实用价值的品种。     

转铁蛋白基因增强水稻对氧化胁迫与稻瘟病菌的抗性

对转豌豆铁蛋白（pea ferritin,Fer）基因水稻T1代的53个株系进行PCR检测,52个株系能扩增出阳性PCR产物。通过测定光合作用过程中最大光化学通量（Fv/Fm值）分析了由百草枯处理引起的T1代水稻叶片的氧化损害。与未转基因水稻相比,转Fer基因水稻的叶片对氧化胁迫的耐受能力有不同程度的增强。百草枯处理后转基因植株叶片叶绿素含量与水处理叶片相比没有明显下降,而未转基因植株叶片叶绿素含量降低至水处理叶片的20％左右。选取9株对氧化胁迫耐受能力较强的水稻进行了Northern blot分析和子代的稻瘟病抗性测定,其中5株转基因植株Fer mRNA 积累增强。病原菌接种后T2代转基因植株的病斑数量明显少于非转基因植株。表明转Fer基因水稻对氧化胁迫和病原菌有较好的抗性。     

小麦转TPS基因植株的获得及其初步功能鉴定

为了探索利用基因工程改良小麦抗旱性和耐盐性的途径,通过基因枪法将具有抗旱和耐盐碱功能的海藻糖合酶(TPS)基因导入普通小麦品种CB9945,获得了转TPS基因的小麦植株,对T0代植株进行了PCR检测,对T2代植株进行了叶片涂抹除草剂检测并进一步进行了验证,鉴定出15个转基因株系.采用模拟抗旱、耐盐环境,对15个转基因株系进行了初步功能鉴定,发现转TPS基因小麦植株的抗旱、耐盐能力得到了一定程度的提高.     

耐盐芦苇大片段BIBAC载体构建及水稻遗传转化

利用盐生植物的耐盐基因改良作物耐盐性是保障土壤盐渍化地区粮食生产和改良盐渍化土地的最经济最有效的途径。本研究以生长于西北盐盖上的一种耐盐野生芦苇为材料,制备了Mb级耐盐芦苇基因组DNA,酶切后经脉冲场凝胶电泳将其分离为不同大小的大片段DNA区段,并与酶切回收的BIBAC-S载体进行连接,成功构建了耐盐芦苇不同大小的大片段DNA-BIBAC载体,经根癌农杆菌EHA105介导,成功地转化了粳稻品种‘中花11’成熟胚愈伤组织,获得了转基因水稻植株。研究表明,不同大小的大片段DNA-BIBAC载体在转化同一受体材料时,其所获得的愈伤转化率、植株转化率、转基因植株的阳性率等存在差异,说明插入片段的大小与转化效率之间存在关系。本研究为创制耐盐水稻新种质、克隆盐生植物的耐盐基因提供了理论和技术参考。     

Salt Tolerance in Rice:Focus on Mechanisms and Approaches

Salt tolerance is an important constrain for rice, which is generally categorized as a typical glycophyte. Soil salinity is one of the major constraints affecting rice production worldwide, especially in the coastal areas. Susceptibility or tolerance of rice plants to high salinity is a coordinated action of multiple stress responsive genes, which also interacts with other components of stress signal transduction pathways. Salt tolerant varieties can be produced by marker-assisted selection or genetic engineering by introducing salt-tolerance genes. In this review, we have updated on mechanisms and genes which can help in transferring of the salt tolerance into high-yielding rice varieties. We have focused on the need for integrating phenotyping, genomics, metabolic profiling and phenomics into transgenic and breeding approaches to develop high-yielding as well as salt tolerant rice varieties.     

Effects of salt and low light intensity during the vegetative stage on susceptibility of rice to male sterility induced by chilling stress during the reproductive stage

We tested whether exposing rice plants to abiotic stress (salt or shade) during vegetative growth affects the chilling tolerance of reproductive organs, which is one of the most important traits for rice growing in a cool climate; we used two rice cultivars with different tolerance in two growing seasons. We divided the vegetative growth into three phases to clarify the most sensitive period: 7–22 days after transplanting (DAT), 23–38 DAT and 39–54 DAT. Chilling tolerance of the pre-stressed plants was based on the male sterility induced by low temperatures. Shade and salt stress during all three vegetative growth phases significantly reduced stomatal conductance. Shade decreased the specific leaf weight and the leaf sugar and starch contents, but salt had no significant effect, despite causing leaf damage. Low temperatures during the reproductive stage induced spikelet sterility in all plants, but the magnitude was greater in the salt- and shade-stressed plants of both cultivars, especially those stressed late during vegetative growth. The increased spikelet sterility caused by chilling was closely related to the reduction of the total spikelet number per panicle. This is the first study to show that salt and low light stress during vegetative growth increased the susceptibility of rice plants to chilling damage during panicle development.     

Stress-Activated Protein Kinase OsSAPK7 Regulates Salt-Stress Tolerance by Modulating Diverse Stress-Defensive Responses in Rice

Soil salinity is an environmental threat limiting rice productivity. Identification of salinity tolerance genes and exploitation of their mechanisms in plants are vital for crop breeding. In this study, the function of stress-activated protein kinase 7(OsSAPK7), a SnRK2 family member, was characterized in response to salt stress in rice. Compared with variety 9804, OsSAPK7-overexpression plants had a greater survival rate, increased chlorophyll and proline contents, and superoxide dismutase and catalase activities at the seedling stage under salt-stress conditions, as well as decreased sodium potassium ratio(Na~+/K~+) and malondialdehyde contents. After salt stress, the OsS APK7 knockout plants had lower survival rates, increased Na~+/K~+ ratios and malomdiadehyde contents, and decreased physiological parameters compared with 9804. These changes in transgenic lines suggested that OsSAPK7 increased the salt tolerance of rice by modulating ion homeostasis, redox reactions and photosynthesis. The results of RNA-Seq indicated that genes involved in redox-dependent signaling pathway, photosynthesis and zeatin synthesis pathways were significantly down-regulated in the OsSAPK7 knockout line compared with 9804 under salt-stress condition, which confirmed that OsSAPK7 positively regulated salt tolerance by modulating diverse stress-defensive responses in rice. These findings provided novel insights for the genetic improvement of rice and for understanding the regulatory mechanisms of salt-stress tolerance.     

转录因子OPBP1和OsiWRKY基因的超表达提高水稻的耐盐及抗病能力

采用基因枪法将耐盐的OPBP1和抗病相关的OsiWRKY转录因子共转化入水稻材料秀水11,经PCR及分子杂交分析确认目的基因已整合到水稻基因组中并超量表达。抗病性测定表明,与未转基因水稻相比,转基因水稻植株表现出对稻瘟病菌（Magnaporthe grisea）和白叶枯病菌（Xanthomonas oryzae pv.oryzae）的较好抗性。抗盐能力检测结果表明,在同等盐胁迫条件下转基因株系生长较快,叶绿素含量和生物学产量都显著高于未转基因对照。上述结果表明,OPBP1和OsiWRKY基因在水稻中增强表达可以提高水稻的耐盐及抗病能力。     

Allelic Analysis for bar Gene from Different Rice Transformation Events

Abstract

Mechanism(s) of gene transformation and integration in rice (Oryza sauva L.) is/are not currently well understood. This research was conducted to determine whether a transgene is inserted into the rice genome specifically or randomly. Seven homozygous transgenic Taipei (T) 309 and Nipponbare plants with the bar transgene from different rice transformation events were crossed. The segregation of F2 and F3 populations from a total of 21 crosses was studied in a greenhouse and field to determine if the genes were allelic or non-allelic. Five genomic locations appeared to be involved among the seven transgenic plants. An additional 20 homozygous transgenic T309 plants, with the bar transgene from different transformation events, were crossed reciprocally with the previous seven plants. One hundred and fifteen crosses made during 1999 and 2000 were analyzed for allelism. In some combinations, the genes were allelic, but most of them were non-allelic, with two or more pairs of genes being expressed. Twenty loci among the 27 transgenic plants were involved and some plants had several inserted genes expressed. Genes in nine out of 27 transformed plants were allelic. We concluded that the functional foreign (bar) gene was restrictively/preferentially inserted into the rice genome in some cases and was not completely randomly inserted and expressed in the rice genome. If the mechanism(s) for preferential insertion were identified, rice researchers could possibly control insertion sites of transgenes to optimize gene expression.     

OsBTF3过量表达和RNAi转基因水稻抗盐和抗低温胁迫鉴定

 为评估水稻OsBTF3基因在水稻对非生物逆境胁迫应答中的功能,利用RT Q PCR技术,对经高盐(200 mmol/L NaCl溶液)和低温(4℃)胁迫处理后水稻的OsBTF3基因表达动态进行了测定,并对前期获得的OsBTF3过量表达和RNAi转基因水稻株系进行了抗高盐和抗低温胁迫的鉴定。结果表明,高盐胁迫处理显著抑制了OsBTF3基因的表达,低温胁迫对其表达影响有一定的变化;转基因T2代过量表达株系对高盐和低温胁迫抗性显著增强,而RNAi转基因株系抗性减弱,说明OsBTF3可能在水稻对高盐和低温胁迫反应中起着重要的调控作用。