Osmotic Stress Resistance of Transgenic Tobacco Expressed Soybean Lysine-rich Protein Gene

PM2 gene （accession number： M80664） with high lysine content from soybean （Glycine max） was found in GenBank by changing three BLASTp parameters. Amino acid composition analysis of PM2 showed that Lys content was on the high level of 18.22%. Protein encoded by PM＇2 also belonged to the family of late embryogenesis abundant （LEA） proteins, which was considered that it had a strong relation with the abiotic stress resistance. In this experiment, PM2 gene was obtained from dry soybean seeds by RT-PCR, plant expression vector pEMTPM2 was constructed, and then transformed into tobacco by using agrobacterium-mediated method. Eight salt and drought tolerant lines were obtained from 31 differentiated lines. Real-time PCR showed that PM2 gene overexpressed in all four PCR positive lines with the osmotic stress resistance. These results confirmed that the overexpression of PM2 gene enhanced the osmotic stress resistance of transgenic tobacco.     

Breeding of Selectable Marker-Free Transgenic Rice Lines Containing AP1 Gene with Enhanced Disease Resistance

In order to obtain marker-free transgenic rice with improved disease resistance, the AP1 gene of Capsicum annuum and hygromycin-resistance gene （HPT） were cloned into the two separate T-DNA regions of the binary vector pSB130, respectively, and introduced into the calli derived from the immature seeds of two elite japonica rice varieties, Guangling Xiangjing and Wuxiangjing 9, mediated by Agrobacterium-mediated transformation. Many cotransgenic rice lines containing both the AP1 gene and the marker gene were regenerated and the integration of both transgenes in the transgenic rice plants was confirmed by either PCR or Southern blotting technique. Several selectable marker-free transgenic rice plants were subsequently obtained from the progeny of the cotransformants, and confirmed by both PCR and Southern blotting analysis. These transgenic rice lines were tested in the field and their resistance to disease was carefully investigated, the results showed that after inoculation the resistance to either bacterial blight or sheath blight of the selected transgenic lines was improved when compared with those of wild type.     

Effect of the Antisense BcMF12 Driven by the BcA9 Promoter on Gene Silencing in Brassica campestris L. ssp. chinensis

The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi （Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis） and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi.     

Production of Transgenic Anliucheng Sweet Orange （Citrus sinensis Osbeck） with Xa21 Gene for Potential Canker Resistance

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR （pattern recognition receptor） gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene （HPT） was introduced into Anliucheng sweet orange （Citrus sinensis Osbeck） via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin （HYG） and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identiifed by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was brielfy discussed.     

Specific Expression of Maize SBEIIb Promoter Mediated by Different Promoter Region in Transgenic Tobacco Plants

Starch branching enzyme （SBE） catalyzes the biosynthesis of amylopectin. We described the isolation and characterization of SBEIIb promoter and their expression patterns in transgenic tobacco. Using the genomic DNA of maize cultivar Lunuo 1 as template, the SBEIIb promoter was isolated by PCR and was cloned into pMD18-T vector. To study SEBIIb gene regulation at the cellular level, SBEIIb promoter was fused to the ~-glucuronidase （GUS） report gene. The results of the fluorometric GUS assays indicate that the sbeⅡb-GUS fusion directed a seed-specific expression. Four series of constructs were made with the promoter and the GUS reporter gene to investigate the cis-acting analysis, showing that the four different constructs all can drive expression of the GUS gene in seed plumule and cotyledon and the GUS activity was apparently decreased with the progressive loss of promoter 5＇ end.     

Transformation of TrxS Gene into Barley by Particle Bombardment

The production of malting barley in China can＇t meet the demand of beer industries because of poor quality and it becomes a bottleneck problem in beer manufacture industry. In this paper, TrxS gene cloned from Phalaris coerulescens was transferred into barley cultivar Yupi 1 （YP1） via biolistic bombardment. 1206 immature embryos were bombarded and seven transgenic plants carrying TrxS gene were confirmed by PCR and PCR-Southern blotting analysis. TrxS gene was expressed in transgenic plants by RT-PCR analysis. The activity of Trxh and α-amylase of transgenic line were higher than that of non-transgenic line, which is helpful to improve malting quality of barley.     

Stability of PAC(Psy-2A-Crt I) gene and agronomic traits in the F_(2:3) of IR36/PAC transgenic plants

This study was carried out to evaluate the expression of yellow endosperm color in the progeny of Psy-2A-Crt I(β-carotene gene, PAC) transgenic plants crossed with IR36. The selected 25 lines of F2 progeny exhibited stable expression of yellow endosperm color and high agronomic characteristics. IR36 and PAC transgenic plant of the grain length, width and ratio showed the same tendency of normal distribution. For progeny selection, colorimeters were as employed to distinguish differing visible colors. Correlation analysis was conducted to investigate the relationship between b* value and the expression of specific genes. b* value was associated with the relative expression of the PAC-Rev and MAR product by real-time PCR and t-test revealed significant difference. Based on this study, correlation between colorimetric values and real-time PCR was effective to detect gene expression. Yellow endosperm progenies represent a new genetic pool that might be useful to increase the genetic diversity of yellow endosperm rice.     

GmSARK启动子驱动IPT基因在拟南芥中的表达研究

[目的]对GmSARK启动子驱动IPT基因在拟南芥中的表达进行研究。[方法]分别克隆IPT基因及GmSARK基因启动子,构建它们的植物表达载体并进行拟南芥的转化,利用PPT(phosphinothricin)除草剂筛选,检测T1代转基因植株;对T1代转基因植株进行黑暗避光和干旱处理后进行半定量RT-PCR分析。[结果]成功克隆得到了IPT基因及GmSARK基因,并构建了它们的p3301-GmSARK-IPT植物表达载体;对T1代转基因植株的RT-PCR表明,目的基因mRNA水平上有所表达。[结论]为进一步研究GmSARK启动子驱动的IPT基因在抗逆中的作用奠定了基础。     

蜡梅SAMT基因在烟草中的遗传转化及其功能分析

为研究蜡梅SAMT 基因的调控功能,利用农杆菌介导法将其转入烟草中。经PCR 方法对转基因烟草进行检 测,结果显示:16 株转化植株中有15 株扩增出了目的条带,进一步的RT-PCR 检测结果表明,阳性植株均发生了正 确转录。对转基因烟草植株和未转基因对照植株进行植株大小、叶片形态、花色、花瓣大小以及花期进行观察,均 未发现有明显差异。采用顶空固相微萃取以及气相色谱-质谱技术(HS-SPME-GC-MS)对转基因烟草鲜花进行花 香成分分析,结果表明,转CpSAMT 基因烟草中有较高含量的苯甲醇、己烯醇、芳樟醇、石竹烯和苯甲醛等成分,但未 能检测到水杨酸甲酯和苯甲酸甲酯成分。      

水杨酸诱导表达AtCBF1的转基因烟草研究

CBF是植物低温驯化过程中的关键性调控因子,过量表达CBF可显著提高转基因植物的非生物胁迫抗性。但是,组成型高表达CBF基因常导致转基因植株生长受阻。通过使用人工合成的水杨酸诱导型启动子,结果发现拟南芥CBF1基因在转基因烟草中受低浓度水杨酸诱导表达,且不影响烟草的生长发育。本研究结果说明,组合化学诱导型启动子和CBF基因,可以用于提高作物的抗环境胁迫的遗传改良中。     

转G10aroA棉花株系的获得及分子生物学鉴定

【目的】培育具有抗草甘膦除草剂的棉花材料具有极其重要的意义。利用农杆菌介导法在棉花中转入编码EPSPS酶的抗草甘膦除草剂基因G10aroA,通过体细胞愈伤诱导组织培养技术获得能够稳定遗传的转基因棉花株系材料。【方法】首先,利用不同草甘膦抗性筛选条件比较分析不同棉花受体材料的愈伤诱导效率;其次,以R15材料作为受体,利用含有G10aroA的农杆菌侵染下胚轴切段,在进行体细胞愈伤诱导的组织培养过程中通过草甘膦抗性筛选获得棉花再生植株,对获得的棉花再生植株进行纯合繁育。在此基础上,利用PCR扩增检测证实外源G10aroA在转基因植株中能够稳定遗传;利用RT-PCR分析其外源基因在转基因植株不同组织中的转录水平进行研究、并进一步利用Western-blot对转基因植株中外源蛋白的表达进行分析。【结果】在优化的草甘膦筛选条件下,以草甘膦浓度为2.5 mmol•L-1的抗性条件进行棉花愈伤诱导筛选并获得棉花再生植株;利用特异引物进行PCR检测结果表明,在检测的全部再生植株中,扩增得到1.8 kb预期大小目标条带的阳性株系32株,其中,收获的27个株系的外源目标基因能够在T0、T1转基因植株中稳定遗传;对G10aroA在转基因株系L12、L14的不同组织中的转录表达进行定量RT-PCR分析表明,外源G10aroA在转基因棉花植株的不同组织中表达具有差异,相对表达量高低依次为茎、苞叶、叶和花;另外,蛋白检测结果进一步表明,该外源基因能够在转基因株系L7、L12和L14中植株中正常表达为预期46 kD的EPSPS蛋白。【结论】通过农杆菌介导法转化外源抗草甘膦基因G10aroA,在草甘膦抗性条件下进行棉花体细胞诱导的组织培养,成功获得转外源G10aroA的棉花再生株系,并通过分子生物学方法研究证实外源G10aroA能够在T0、T1转基因株系中稳定遗传、转录以及表达。     

利用RNAi技术抑制籽粒PPO合成改良小麦面粉白度的研究

【目的】通过RNAi策略抑制小麦籽粒ppo的表达,获得籽粒PPO酶活性低、白度高的转基因小麦新种质。【方法】构建了以小麦胚乳特异表达启动子1Dx5启动子驱动的籽粒ppo的RNAi载体pBAC47P-ppoIR,利用基因枪将该载体与含bar的表达载体基因枪共转化中优9507幼胚愈伤组织,获得T0转基因植株。通过分子鉴定（PCR、Southern杂交、半定量RT-PCR、Northern杂交）、酶活筛选和白度测定等方法对转基因植株及其后代株系进行筛选。【结果】获得了27株阳性T0转基因植株。转基因植株及其后代经PCR和Southern杂交检测表明,RNAi构件已经稳定整合到T1的12个株系中。半定量RT-PCR和Northern杂交结果表明有20株T2籽粒中ppo表达水平明显低于对照。对转基因T2灌浆期籽粒进行PPO同功酶活性分析表明,有6个株系的PPO活性有所减弱。对T4籽粒的面片白度测定表明,5个株系的白度均比对照有所提高,其中2个株系效果显著。【结论】RNAi技术的表达能抑制籽粒ppo表达,降低PPO同功酶的活性,明显提高小麦面片白度,从而为小麦面粉白度的改良、品质育种提供了材料。     

东莞大蕉超表达拟南芥CBF1基因及其抗寒性检测

【目的】研究超表达拟南芥CBF1基因（AtCBF1）对大蕉抗寒性的影响,为从大蕉克隆抗寒相关基因奠定基础。【方法】采用农杆菌介导法转化东莞大蕉的胚性细胞悬浮系,获得转AtCBF1基因的大蕉植株;利用GUS组织染色、PCR、RT-PCR以及RT-qPCR对转基因植株进行鉴定;比较低温处理后的转基因株系和对照的冷害特征以及超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量等生理生化指标,鉴定转基因大蕉植株的抗寒能力。【结果】试验共获得6个抗性再生转化系。GUS组织染色结果表明,除T1外,其余均为阳性;PCR鉴定结果表明,AtCBF1在6个抗性再生转化系均为阳性,而GUS基因在T1转化系中没有检测出;RT-PCR结果表明,AtCBF1在6个转化系均得到表达,对T1、T2和T3 3个转化系进行RT-qPCR检测发现,AtCBF1基因在3个转基因株系表达水平存在差异;在低温处理下,转基因植株的叶片相对电导率、MDA的累积都低于非转基因植株,而SOD总活性高于对照;低温处理条件下,转基因植株叶片的冷害症状明显轻于对照。【结论】AtCBF1在大蕉中超表达,具有增强大蕉SOD活性,降低因低温导致的MDA含量和离子渗漏率,缓解质膜过氧程度,进而改善大蕉植株抗低温胁迫的能力。     

棉花蔗糖合成酶3基因第1个内含子在基因表达中的作用

为了研究棉花SUS3基因编码区的第1个内含子在基因表达调控中的作用,分离该内含子并将其插入到GUS基因编码区中构建载体Susfig::121,将载体Susfig::121通过农杆菌介导转入拟南芥.对转基因拟南芥进行GUS活性分析,发现该内含子对GUS基因在拟南芥中的表达具有负调控作用,并且特异抑制报告基因在花粉中表达....     

水稻启动子OsN1p的克隆与功能分析

将OsN1基因上游881 bp启动子(OsN1p)序列取代pBI121中gus基因上游的35S启动子,构建植物表达载体pBIN1p,经农杆菌介导转化水稻品种‘日本晴’,获得转基因植株。GUS组织化学染色结果表明,由该启动子驱动的gus基因能在愈伤组织中较低水平地表达;稻瘟病菌接种转基因植株24 h后,GUS活性为未接种前的4.2倍;5 mmol.L-1水杨酸(SA)和0.5 mmol.L-1茉莉酸甲酯(MeJA)分别喷施转基因植株叶面6 h后,GUS活性分别为处理前的5.9和2.4倍。表明,OsN1p启动子具有启动活性,同时明显具有受稻瘟病菌、SA和MeJA诱导表达的特性。     

蕙兰Flowering locus T基因的克隆及其对开花的影响

【目的】 探讨CfFT基因在蕙兰成花中的作用。【方法】采用RT-PCR结合RACE技术从蕙兰(Cymbidium faberi)中克隆Flowering locus T（FT）同源基因CfFT。采用实时定量RT-PCR对不同组织及不同花发育时期CfFT进行表达分析。将该基因克隆到PBI121载体上导入烟草中,并对不同转基因烟草株系中的CfFT、NFL、NtFUL和NAP1基因进行实时定量RT-PCR分析。【结果】对蕙兰花芽分化不同时期的CfFT的表达分析表明,CfFT在花芽分化初期的表达量最高,之后随着花芽的成熟表达量逐渐降低。将CfFT导入烟草进行异源表达,转基因株系表现出明显的早花表型。对开花时间不一的转基因株系中的CfFT表达分析表明,其表达量与转基因烟草开花时间早晚成正比。进一步对这些株系内源的NFL、NAP1和NtFUL表达分析表明,NFL、NAP1和NtFUL基因的表达量与CfFT表达成正比,说明NFL、NAP1和NtFUL的表达受FT基因的上游调控。【结论】在烟草中异源表达蕙兰中的CfFT基因能促进烟草提前开花。