TRANSFER OF THE ATRAZINE-RESISTANT GENE OF BLACK NIGHTSHADE TO SOYBEAN CHLOROPLAST GENOME AND ITS EXPRESSION IN TRANSGENIC PLANTS

The atrazine-resistant psbA gene of black nightshade was transferred to the chloroplast genome of atrazine-susceptible soybean by means of ovary microinjection during the stage of zygote. The identification was carried out by using the methods of spraying the leaves directly with atrazine solution, examining the change of leaf fluorescence kinetics under a brighter light induction, molecular hybridization, etc. The experimental results show that the transgenic soybean plants do have been obtained for the first time.

Development and identification of glyphosate-tolerant transgenic soybean via direct selection with glyphosate

Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD_(600) of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T_0 transgenic plants, consisting of spotting plantlets with 1 μL Roundup~?. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T_0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.

Specific Expression of a Novel Nodulin GmN479 Gene in the Infected Cells of Soybean (Glycine max) Nodules

A novel nodulin gene, GmN479 genomic clone composing of 3 630 nucleotides was isolated from mature soybean nodules using GmN479 cDNA as a probe by subtractive hybridization procedure. GmN479 encodes 170 amino acids with 2.09 kb nucleotides promoter region, and contains two important upstream promoter elements, one is a conserved cis-acting sequence motif 5′-AAAGAT-3′ controlling nodulin gene expression, and the other is typical CAAT boxes. GmN479 gene has a single zinc-finger C2H2 domain YSCAFCQRGFSNAQALLGGHMNIH and a conserved motif, QALGGHMN in the zinc-finger with a short leucine repeat in the LDLELRLGL motif closed to C-terminal. These two conserved motifs share respectively higher identity with those in the floral regulator SUPERMAN gene, indicating that GmN479 may function as a transcriptional regulator, and is a likely candidate for playing a role in nodule-morphogenesis. Blotting data showed that GmN479 is a single copy presenting in the genome of soybean nodule, and its expression profile is similar to that of Lb-a, but it is different from that of ENOD2. GUS staining showed that GmN479 promoter just functions in the infected cells of nodules, indicating that the GmN479 is one of the truly symbiotically induced host genes, and belongs to a late nodulin gene. The expression pattern of GmN479 gene seems to imply that it may be closely related to the development of the nodule. In a sense, it may be a useful marker for identifying the development of the infected cell system in the nodules of soybean.

Stable expression of Arabidopsis vacuolar Na^+/H^+ antiporter gene AtNHX1,and salt tolerance in transgenic soybean for over six generations

The Arabidopsis vacuolar Na+/H+ antiporter gene,AtNHX1,was introduced into soybean by Agrobacterium-mediated transformation.Four independent kanamycin resistant lines were obtained.The result of PCR,Southern blotting and Northern blotting analyses demonstrated that the AtNHX1 gene was successfully inserted into the soybean genome and stably expressed in these kanamycin resistant lines.The stability of AtNHX1 expression and salt resistance were evaluated in the soybean transformants for over 6 generations.Two independently derived transgenic lines with high expression level of AtNHX1 were selected,and propagated to generation T5 in the absence of selection pressure.PCR and RT-PCR examinations revealed that AtNHX1 was highly expressed in all investigated transgenic T5 progenies.Furthermore,all transgenic T5 plants showed resistant to salt stress,same as those of homozygous T2 plants.Taken together,our results indicated that constitutive expression of AtNHX1 enhanced salt tolerance in soybean for over 6 generations,suggesting a great potential use of AtNHX1 for improving salt tolerance in plants by genetic engineering.

抗阿特拉津(Atrazine)转基因大豆植株的田间检测表现

地面(出苗前)或植株(幼苗分枝期)喷施Atrazine液后,从田间观测及考种结果看,未导入抗性基因的大豆对照株受害严重,或枯死或严重减产;导入抗性基因的大豆植株后代F_4、F_5代基本不受影响,单株产量与未喷液的对照株相近或略高。这显示了导入的抗性基因对Atrazine的抵抗作用。通过喷施Atrazine已筛选出了多个抗性较强的株系,有望从中进一步筛选出抗性稳定的株系,用到大田生产。

抗atrazine转基因大豆的抗性遗传及某些生理、农艺性状

抗ａｔｒａｚｉｎｅ基因导入大豆植株后不仅在Ｆ１代获表达，且已遗传到后代（１９９年已至Ｆ７代）。通过正交与反交试验证明，其Ｆ１代植株对ａｔｒａｚｉｎｅ的抗性性状均与其母本性状一致，即该性状为母系遗传，为细胞质基因，位于叶绿体ＤＮＡ上。通过导入抗性基因株与未导入抗性基因的对照株的光合速率测定、成分分析及农艺性状（株高、百粒重、单株产量）测定表明，两类植株间无明显差异。在喷施ａｔｒａｚｉｎｅ情况下，转基因株能表现出抗性。

Isolation and functional analysis of transcription factor GmWRKY57B from soybean

WRKY proteins are a large family of transcriptional regulators in higher plants involved in many biological processes, such as responses to biotic and abiotic stress, plant development and metabolism. In this study, a soybean cDNA library was constructed and screened by yeast one-hybrid system using the 3×W-box element as a bait, and consequently a transcription factor, named GmWRKY57B, a member of group 3 of WRKY family, was obtained. GmWRKY57B specifically binds to the W-box of the Arabidopsis NPR1 (AtNPR1) promoter in yeast cells. This specific binding was further confirmed with an in vitro electrophoretic mobility shift assay. RT-PCR analysis showed that GmWRKY57B was expressed at a similar level in root, stem and leaf of the soybean plant. Overexpression of the GmWRKY57B gene in tobacco conferred the transgenics tolerance to drought stress. This result indicated that the GmWRKY57B gene plays a role in plant tolerance to abiotic stress.

分散固相萃取超高效液相色谱串联质谱法同时检测大豆植株及土壤中烟嘧磺隆、莠去津及其代谢物残留

土壤中烟嘧磺隆和莠去津的高残留往往会导致后茬大豆药害问题。本研究建立了一种分散固相萃取超高效液相色谱串联质谱法同时检测大豆植株及土壤中烟嘧磺隆、莠去津及其代谢物残留方法。样品经含2%甲酸的乙腈提取,经分散固相萃取净化,以乙腈和0.2%甲酸水为流动相,采用Poroshell 120 EC-C18色谱柱梯度洗脱,基质匹配标准曲线外标法定量分析。结果表明:在0.01、0.10 mg/kg和1.00 mg/kg添加水平下,烟嘧磺隆、莠去津及其代谢物的平均回收率为70%~113%,相对标准偏差为0.3%~11.8%,目标化合物质量浓度与对应的峰面积之间在0.001~1 mg/kg范围内线性关系良好,决定系数R^(2)≥0.9841,方法的定量限为0.01 mg/kg。该方法简便、快捷、准确,适用于土壤和幼苗期至鼓粒期大豆植株中烟嘧磺隆、莠去津及其代谢物的检测。本研究为烟嘧磺隆和莠去津的科学使用及后茬作物的安全种植提供有效监测方法。

通过农杆菌介导法将Bt(cryIA)基因导入大豆

采用农杆菌介导的大豆子叶节转化系统成功地将Bt基因 (cryIA)导入大豆。从发芽 5 - 7天的大豆无菌苗切取子叶节外植体 ,经农杆菌感染和共培养后 ,在选择培养基上 4周左右出现抗性不定芽。将不定芽转移到芽伸长培养基上 ,4 - 6周后再生苗长至 2 .5 - 3cm高。再将再生苗切下转入生根培养基 ,2周左右生根。生根后的再生植株经逐步锻炼移入盆中 ,所有植株均能正常开花结荚。在移栽成活的 8株T0 植株中有 7株PCR检测呈阳性反应 ;在 7个T1株系中有 4个株系存在PCR阳性植株。取 4个稳定遗传的T1代株系内的阳性植株的叶片提取DNA ,用地高辛标记的Bt基因探针进行Southern杂交分析 ,结果 4个株系均呈现阳性 ,证明Bt基因已整合到受体大豆的基因组内并能传递给后代。

改良大豆子叶节再生体系的研究

采用大豆种子萌发5—6d后的子叶节作外植体，对农杆菌介导的大豆遗传转化系统及其影响因素进行r研究。结果表明，将子叶节与农杆菌共培养60h后放入含卡那霉素50mg／L的诱芽培养基上，待苗长至4-5cm后放入生根培养基中进行生根，最后移栽到培养土中，效果较好。对大豆遗传转化再生的主要因素，如大豆基因型、诱导出芽所需激素浓度、卡那霉素筛选压力等条件做了一系列的研究，建立了一个比较好的遗传转化系统，其转化率达到2．8％。

抗阿特拉津转基因大豆植株后代的遗传分析

本试验用阿特拉津溶液涂抹、荧光诱导动力学检测、分子杂交等方法对抗阿特拉津转基因大豆植株的后代进行了鉴定,在第二代及第三代中检测到了抗性基因的存在,表明从龙葵中得到的此抗阿特拉津 psbA 基因不仅能导人大豆叶绿体基因组中获得表达,而且可以遗传到后代。

苏云金芽孢杆菌杀虫晶体蛋白基因导入大豆的研究

用苏云金芽孢杆菌(BacillusthuringiensisBerliner)杀虫晶体蛋白(Bt)基因和葡糖苷酸酶(GUS)基因通过基因枪轰击和根癌土壤杆菌(Agrobacteriumtumefaciens(SmithetTownsend)Conn)介导转入大豆(Glycinemax(L.)Merr.),诱导大豆转基因植株再生。大豆主栽品种“中黄4号”和品系8502未成熟子叶有较强体细胞胚分化能力。体细胞胚的脱水处理显著促进“中黄4号”体细胞胚的萌发。未成熟子叶的预培养有利于根癌土壤杆菌感染子叶外植体体细胞胚的分化。基因型和受体的选择,转基因体系的改进,体细胞胚的脱水处理等是提高大豆转基因效率的重要因素。

转hrpZpsta抗病基因大豆的研究

【目的】将具有广谱抗病性的hrpZpsta基因导入大豆,为培育抗灰斑病的转基因大豆新品系奠定基础。【方法】采用农杆菌介导法,以大豆子叶节为受体,将具有广谱抗性的hrpZpsta基因转入大豆品种"吉林30"中,以耐盐基因badh作为筛选标记性基因,经过抗性筛选,对转基因植株进行PCR检测、Southern杂交和RT-PCR检测分析。【结果】确定的NaCl筛选浓度为200mmol/L。对T1、T2和T3代转基因植株进行PCR检测,得到T1代阳性植株30株,T2代45株,T3代284株,说明外源hrpZpsta基因在转基因后代中能够遗传。Southern杂交结果表明,外源目的基因hrpZpsta已经整合进大豆基因组中,且整合位点不尽相同。RT-PCR结果表明,hrpZpsta基因在受体大豆中获得表达。【结论】获得了hrpZpsta基因遗传表达的T3代转基因大豆株系。

转HAL1基因大豆侧翼序列分析及特异性检测方法研究

为方便HAL1转基因大豆的研究和检测,以转HAL1基因大豆T3代基因组DNA为模板,使用热不对称交错PCR(TAIL-PCR)方法分离其外源基因插入位点的侧翼序列,获得了插入片段DNA序列(T-DNA)的左翼序列和右翼序列。通过比对大豆基因组序列确定其整合位点,确定了HAL1基因的T-DNA在大豆基因组1号染色体非编码区49468395位点以单拷贝插入,转基因事件不影响大豆基因组功能基因的正常表达,而且在200 mmol·L^(-1)NaCl盐胁迫下转基因植株生长力明显强于对照材料。蛋白定量检测结果表明,在叶片中蛋白含量最高可达0.03 mg·g^(-1),在根茎花中也有表达,但是含量较低。根据整合位点处的左右侧翼序列设计两对特异性PCR检测引物,PCR检测结果显示只有转HAL1基因大豆阳性材料才能扩增出926和816 bp DNA片段。本研究建立的转HAL1基因大豆特异性定性检测方法,对转基因大豆的研究具有重要的意义,也为大豆转基因受体材料的管理与检测提供参考。

转基因大豆DNA检测芯片的研究

为提高对转基因大豆的监督检测能力 ,研制了转基因大豆DNA检测芯片。根据转基因大豆 (RoundupReady)中所转入的外源基因 ,选择CaMV35S启动子、NOS终止子、NOS EPSPE基因和内源Lectin基因设计特异性引物 ,采用多重PCR法对待测样品进行扩增 ,通过缺口平移法合成DIG dUTP标记杂交探针 ,并制备基因芯片。在对PCR反应和扩增产物与芯片杂交条件进行优化的同时 ,比较了芯片检测的特异性和重复性 ,并对检测的灵敏度进行测试。结果表明 ,该方法具有较好的特异性和重复性 ,检测灵敏度可达 0 5 % ,由于采用了多重PCR技术 ,一次可同时检测多个基因 ,提高了检测的准确性和效率。

胆碱磷酸转移酶基因转化大豆的初步研究

胆碱磷酸转移酶基因(cpt)是磷脂合成代谢途径上一个关键的酶基因,其基因产物是磷脂酰胆碱(PC),PC做为主要膜脂与膜的流动性和植物的抗寒性有密切关系。本实验用花粉管通道法将质粒PRDH401(含35S-35S-AMV-cpt-NOST)导入五个黑龙江常见栽培大豆品种中,对D1 代植株进行卡那霉素抗性筛选、低温筛选和PCR检测,初步筛选到转化植株。

HrpZpsta基因植物表达载体的构建及其在大豆中的转化

利用PCR技术扩增含有hrpZpsta基因的克隆载体pMD18-T-hrpZpsta,以植物表达载体pBI121为基础,将PCR产物插入到此植物表达载体中。利用农杆菌介导的大豆子叶节转化法将构建好的表达载体导入大豆品种吉农28中,选用筛选条件为100mg·L-1的卡那霉素选择培养基培养,对获得的转基因植株进行PCR检测,结果从部分抗性植株中扩增出hrpZpsta基因,初步证明hrpZpsta基因成功转入到受体大豆中,获得了转基因阳性植株。

高山被孢霉Δ^6-脂肪酸脱氢酶基因转化大豆的研究

目的从高山被孢霉中克隆的Δ6-脂肪酸脱氢酶基因与植物表达载体pBA121连接,构建重组质粒pBMACL6,采用农杆菌介导的大豆子叶节转化系统成功地将该基因导入到栽培大豆吉林47品种中,获得一批转基因植株。方法利用子叶节外植体诱导出丛生芽和再生植株,利用卡那霉素的抗性筛选培养基连续筛选以及PCR方法、DNA分子杂交分析检测转基因植株中的Δ6-脂肪酸脱氢酶基因表达状况。结果经PCR检测和DNA分子杂交分析,初步证明Δ6-脂肪酸脱氢酶基因已导入并整合到大豆基因组中。结论成功地将Δ6-脂肪酸脱氢酶基因导入并整合到大豆基因组中。

大豆microRNA168调控植物低磷胁迫响应

为研究miR168在大豆响应低磷胁迫中的调控作用,从大豆中克隆了编码大豆miR168的基因MIR168,连接到植物表达载体后转化烟草,获得超量表达阳性转基因株系后鉴定其对低磷胁迫的耐受性。研究结果表明,超量表达MIR168烟草在低磷胁迫时长势强于对照,根长较对照长,地上部和地下部鲜重也显著高于对照,表明MIR168能够提高烟草对低磷的耐受性。MIR168过量表达烟草中miR168表达高于对照,其推定的靶基因AGO1的表达高于或与对照相当,表明miR168和AGO1之间存在相互调控。进一步分析miR168参与的信号调控发现,超量表达MIR168的烟草种子萌发受ABA和JA抑制,对GA3不敏感,表明miR168可能参与了ABA和JA信号调控。本文研究结果将有助于应用大豆miR168调控植物低磷胁迫,为培育磷高效植物提供基因资源。

转基因大豆Roundup Ready调控元件在食品加工过程中降解变化的研究

35S启动子可能会通过基因的水平转移插入到某一致癌基因上游 ,活化并导致癌症的发生。为了解转基因植物中调控元件的安全性问题 ,以转基因大豆RoundupReady为实验材料 ,针对Roundupready转基因大豆中CaMV35S启动子及NOS终止子的序列 ,设计了不同长度片段的引物 ,通过对CaMV35S启动子和NOS终止子的PCR扩增 ,研究了豆腐、豆奶、豆粉 3种大豆加工食品中磨浆、煮浆、调配、均质、杀菌、喷雾干燥等关键工艺对RoundupReady大豆中调控元件的影响。结果表明调控原件在食品加工过程中的降解变化与其所处位置有较大关系。扩增长度相近的 2个片段 ,包含大豆基因组DNA序列的片段受加工过程的影响较小 ,在 3种豆制品的所有加工过程中均能检测到。而只包含CaMV35S启动子序列的片段仅能在原料中检测到 ,原料经过磨浆后 ,片段大小降至 2 0 0bp以下。NOS终止子受食品加工工艺的破坏和影响较小 ,在被检测食品的每一个加工过程中都能够检测到NOS终止子片段。