The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.).

Transfer of T-DNA from Agrobacterium tumefaciens and A. rhizogenes to cells of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is demonstrated following the inoculation of immature embryos and immature embryo-derived callus. Agrobacterium T-DNA vectors containing the C1/Lc anthocyanin-biosynthesis regulatory genes, the gusA gene or a synthetic green fluorescent protein gene (sgfp-S65T) were constructed from original binary vectors. The visual T-DNA markers were used as cell-autonomous reporters of early Agrobacterium-mediated transformation events in the wheat and barley cells. This localization of the transformed cells revealed a non-random distribution throughout each embryo and callus piece.     

蓝莓VcMYB启动子克隆及其功能初步分析

为探究VcMYB启动子在转录过程中如何发挥调控作用,利用FPNI-PCR法从蓝莓中克隆到调控原花青素合成相关的转录因子VcMYB的768 bp启动子序列。用PLACE和Plant CARE在线启动子预测工具分析了该启动子,结果表明其序列中存在启动子的基本元件CAAT-box和TATA-box,还包含一系列的响应元件,如光响应元件、低温响应元件、防御与胁迫响应元件和茉莉酸甲酯响应元件等。为进一步分析该启动子的功能,构建了该基因启动子与GUS基因融合的植物表达载体VcMYBpro::GUS,并用农杆菌转化拟南芥。对转基因拟南芥进行GUS组织化学染色分析,结果表明该VcMYB启动子能驱动GUS基因在转基因拟南芥中表达,并且经脱落酸(ABA)、4℃低温、LED光照和持续光照处理后,转基因拟南芥中GUS的表达活性增强,推测该基因受ABA、低温和光的调控。     

豌豆终止子rbc-T在转基因小麦研究中的应用

目前常用的转基因载体元件大多为真菌或细菌来源,引发对转基因食品安全性的担忧。本研究克隆了豌豆终止子rbc-T,并用其替换农杆菌终止子nos-T构建转化载体,利用基因枪法将携带Ubi启动子、GUS基因和终止子rbc-T组成的最小表达框片段转化普通小麦,同时以带有nos-T终止子的载体为对照,经过PCR检测筛选获得T2代稳定遗传的转基因小麦株系。GUS组织化学染色及酶活定量分析结果表明,带有nos-T及rbc-T的转基因小麦中均能检测到GUS基因不同程度的表达。因此认为,豌豆终止子rbc-T可以代替农杆菌终止子nos-T用于小麦的转基因研究。     

花粉介导法获得油菜转基因植株研究

以甘蓝型冬油菜品种晋油7号为受体，载有GUS基因的质粒pBI121为供体，在7.5％的蔗糖等渗溶液中，通过花粉介导转化方法将GUS基因导入油菜花粉，随着花粉管的萌发进入胚囊，参与有丝分裂，将外源质粒转入受体材料。经田间植株性状比较、GUS组织化学定位检测、PCR扩增检测及PCR-Southern杂交检测，证明外源GUS基因已整合到油菜基因组中。     

GUS基因分泌型植物表达载体的构建及对烟草的转化

为了验证PR1B分泌肽对谜底蛋白的分泌和增强功能,以GUS基因为靶基因,成功构建了PR1B 和GUS融合基因的植物表达载体,通过三亲融合法转入LBA4404农杆菌,并对烟草进行了转化,在216株卡那抗 性植株中共获得2株GUS染色阳性植株。     

紫羊茅高频植株再生系统的建立与GUS基因的转化

以紫羊茅的成熟种子为外植体材料,研究了2,4-D、有机物及光照对愈伤组织诱导发生、生长状态及绿苗分化能力的影响.结果表明,3～5 mg/L 2,4-D为愈伤组织诱导和继代培养基的适宜浓度,诱导率为46.6%～57.9%,并使愈伤组织保持胚性状态.在继代培养基中添加脯氨酸和天门冬酰氨,并且每培养1～2个月给予15 d左右的散射光培养,使培养了2年多的愈伤组织仍保持99%以上的绿苗分化率.利用基因枪将GUS基因转入继代2年多的愈伤组织,通过组织染色法检测到了高频率的GUS基因瞬时表达.     

Comparison of Different MARs(Matrix Attachment Regions)Effect on Transgene Expression

Three MARs(matrix attachment regions)fragments were cloned from tobacco (Nicotiana tabacum)(MAR1),yeast(Saccharomyces cerevisiae) (MAR3)and kidney bean(Phaseolus vulgaris) (MAR5)which ranged 984,822 and 782 bp,respectively.Sequence analysis showed that all the fragments had fairly high A/T content (73,62 and 75%,respectively),harbored different number and different type of some characteristic motifs of MARs,such as A-box and T-box,etc.The results of in vitro binding assay showed that the three MARs fragments derived from different organisms could bind specifically to the matrix extracted from the tobacco nuclei with different strength,which also demonstrated that these MARs fragments are functionally conserved during evolution.By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-barMARs into tobacco through Agrobacteriummediated procedures,the effects of MARs sequences on the expression of transgenes in tobacco were investigated and compared.The GUS activity in individual transformants showed that,comparing to the controls without additional MARs,the overall transgene expression level in transformants with MARs had been greatly increased while the variations in transgene expression among transformants were decreased in different degrees.In accordance with the results of sequence analysis and in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overall expression level.     

水稻4CL基因启动子引导的GUS在紫外诱导下的组织特异性表达

 4-香豆酸辅酶A连接酶（4CL）是位于苯丙烷类共同代谢途径中类黄酮、木质素以及肉桂酸酯等分支代谢途径交叉点上的关键酶，对整个代谢过程起着重要的调控作用。4CL基因的表达可被真菌侵染，紫外辐射，机械损伤等外界刺激所诱导。笔者将水稻R4CL-1基因启动子与GUS基因融合导入烟草基因组中，并在紫外诱导下对转基因植株进行了β-Glucuronidase活性的组织化学染色分析。在转基因烟草植株幼叶的叶脉及其周围细胞、幼根的根尖细胞及维管组织、成熟花的花瓣及柱头、未成熟种子的表皮细胞和成熟种子的胚中都检测到了GUS活性，而在完全伸展的老叶、茎（幼茎和成熟茎）、老根和花的其它组织中均没有观察到GUS酶活性，从而证明了R4CL-1启动子能在植物发育过程中指导GUS报告基因的组织特异性表达。     

TMV载体上发生的的前体mRNA基因剪接效应

根据以往的报道,TMV基因只存在于细胞质中且不发生基因剪接,前体mRNA(Pre-mR鄄NA)的剪接只能发生在细胞核中。本研究应用RT-PCR,DNA序列测定及GUS INTRON的点突变和荧光检测等研究手段,首次发现TMV载体中GUS基因的表达和前体mRNA的剪接同时发生,证明了GUS基因在TMV载体上的剪接效应。     

水稻rbcS启动子的克隆及其在转基因水稻中的特异性表达

为将高效特异的启动子用于转基因水稻研究,利用PCR技术从水稻‘中花11’基因组DNA中克隆了rbcS启动子,序列分析表明,扩增片段(2 746 bp)与已报道的该基因序列相应区域的同源性达99.2%。将rbcS启动子与GUS报告基因融合构建了由rbcS启动子引导GUS基因的植物表达载体,经农杆菌介导法导入到水稻中。对转基因水稻植株中GU S活性的定性与定量测定结果表明,rbcS启动子可驱动GUS报告基因在转基因水稻植株叶片中的特异性表达,其表达水平高于C aMV 35S组成型启动子,而在转基因水稻植株根和种子等器官中不表达或表达活性极弱,表现出明显的组织特异性。