谷子和甘蔗C4型磷酸烯醇式丙酮酸羧化酶基因的克隆及其转基因水稻光合效率的提高

水稻是我国重要的粮食作物之一，它是一种典型的C3植物。与其它C3作物不一样的是，水稻的生长需要相对较高的温度和充足的阳光照射。然而高温和高光强的生长环境更加适合于C4植物的生长，更加有利于发挥C4植物高光合效率的特点。因此本论文希望将C4植物中固定CO2的酶磷酸烯醇式丙酮酸羧化酶基因导入水稻，获得一种更加适合高温和高光强生活环境的“C4型”水稻，这对于提高水稻的产量，满足人口增长对粮食需求具有重大意义。 本论文从C4植物谷子和甘蔗中克隆了其C4型磷酸烯醇式丙酮酸羧化酶cDNA基因，获得了具有自主知识产权的基因克隆，并将它们导入粳稻品种中花8号，进而对转基因材料的光合生理特性进行了研究。结果如下： 首次从谷子中得到了ppc基因两个cDNA克隆，分别命名为Mppc1和Mppc2。前者是一个C3型的ppc基因，它可能属于在根中特异表达的C3-2型ppc基因;后者是在绿色叶片中大量表达的C4型ppc基因。它们所编码的蛋白的氨基酸残基数分别为961和964，序列同源性为82.5%。C4型PEPC多出的3个氨基酸位于N末端。利用RACE的方法我们得到了谷子C4型ppc基因完整的cDNA序列，包括63bp的5'非编码区，2895bp的编码区和256bp的3'非编码区。 首次获得了甘蔗C4型ppc基因完整的cDNA序列的克隆，命名为Sppc。它包括95bp的5'非编码区、2886bp的编码区，和224bp的3'非编码区。 利用所克隆的基因，分别连上强组成型启动子Ubiquitin启动子和强光调控启动子Rubisco小亚基启动子后，再插入两个标记基因不同的表达载体pCB和pPCB的多克隆位点中，构建了八个含有外源ppc基因的植物表达载体pCB-Pubi-Mppc、pCB-Pubi-Sppc、pCB-PrbcS-Mppc、pCB-PrbcS-Sppc、pPCB-Pubi-Mppc、pPCB-Pubi-Sppc、pPCB-PrbcS-Mppc和pPCB-PrbcS-Sppc。再加上含有玉米完整的C4型ppc 核基因的载体pCB-ZMppc，共有9个载体。利用农杆菌介导法进行了水稻的转化，各个载体都获得了大量的转基因植株。对标记基因潮霉素磷酸转移酶基因hpt和磷酸甘露糖异构酶基因pmi以及导入的目的ppc基因的PCR扩增检测，结果显示绝大多数转基因植株都能扩增出目的片段，而未转化的植株则没有扩增产物。对部分转基因水稻的Southern和Western杂交以及RT-PCR分析都表明，无论从DNA水平、mRNA水平，还是从蛋白质水平上都证明外源ppc基因都成功地导入了水稻，并获得了正确的表达。 对各载体转基因植株PEPC活性大规模的测定表明，转入玉米完整C4型PEPC核基因（有内含子）的水稻表现出极大的表达效率，大多数转基因材料的PEPC活性为对照的10-20倍，其活性最高可达到对照的44倍。转入谷子和甘蔗PEPC基因cDNA的水稻，表达的效率很低，多数材料活性增加仅为对照的2-5倍，但也有极少数材料活性增加了10倍以上。用Rubisco小亚基启动子控制的ppc基因在水稻的表达活性要略高于Ubiquitin启动子控制的ppc基因。以上结果说明ppc基因的内含子在其转录或mRNA的稳定上起着重要作用。 对部分转基因材料气体交换特征的研究发现，随着转基因水稻PEPC活性的增加，净光合速率也有逐渐增加的趋势。其中PEPC活性最大的ZM24株系的三个单株净光合速率比对照增加了39.8%、13.7%和28.6%，而它们的PEPC活性比对照分别增加了21.2、21.9和23.6倍。 转PEPC水稻的净光合速率与气孔导度具有显著的相关性。这说明表达的外源ppc 基因产物PEPC参与了转基因水稻的气孔运动，使气孔开放程度增加。更有意义的是过表达PEPC的水稻具有更高的水分利用效率，这就增加了其耐旱能力。在光抑制条件下转基因水稻也具有更高的光合能力。这些特征表明转ppc基因的水稻比对照更加适合于水稻高温高光强和干旱的原生环境。

植物体内谷胱甘肽还原酶的功能研究

谷胱甘肽还原酶（GR，EC1.6.4.2）是一重要的抗氧化酶，许多生理学和遗传工程研究都证明GR酶在抗氧化中的重要作用。但改变GR酶怎样影响植物的抗氧化系统却不清楚。GR是抗坏血酸－谷胱甘肽循环途径中的重要组成部分，其功能必然与其密切相关。本文用RNAi技术获得具有较低GR酶活性的转基因烟草，系统测定了非胁迫条件和胁迫条件下抗坏血酸－谷胱甘肽循环的变化，得出以下主要结果： 1．选择一烟草叶绿体GR酶编码基因（X76293, gi: 431954）进行RNAi载体构建，构建好的双元载体转化根癌农杆菌LBA4404，然后侵染转化烟草叶圆片。获得的转基因烟草具有30－70％的GR酶活性。分子检测结果表明GR在RNA和蛋白水平上与GR酶活性的变化一致。文中我们第一次用2-D电泳对烟草中GR同工酶进行分析，并确定发生抑制的GR同工酶在细胞中的定位。2-D电泳后的Western杂交检测到烟草的10种GR同工酶，pI值分布在4.5-6.3，其中3种GR同工酶定位在叶绿体内，其蛋白量占据所有GR酶含量的大部分。RNAi发生在叶绿体内和叶绿体外，表明发生抑制的GR同工酶的基因序列具有很高的同源性。igr转基因烟草在表型上与野生型对照烟草无明显差异。 2．所有igr转基因植株和对照植株中的活性氧（O2-和H2O2）、MDA含量和光合作用都无明显差异，表明正常生长条件下GR酶活性的降低不会引起氧化胁迫。测定正常生长条件下igr转基因烟草中谷胱甘肽库的变化。结果表明与对照烟草相比，GR酶活性降低70％会引起转基因植株中GSH/GSSG比率明显降低，而GSH和GSSG的含量稍有增加；测定抗坏血酸－谷胱甘肽循环的变化，结果显示igr转基因烟草中DHAR和MDHAR的酶活性升高，表明非胁迫条件下较低的GR酶活性可能会诱导抗坏血酸－谷胱甘肽循环不能正常的运转。这一作用可能与改变的谷胱甘肽库有关。GR酶活性降低30％的转基因烟草中未检测到这些变化，表明70％的GR酶活对于非胁迫条件下igr转基因烟草可能是足够的。 3. MV处理结果显示，igr转基因烟草的离体叶圆片和活体植株在MV处理后都发生比对照烟草严重的光漂白作用。igr转基因烟草的活性氧和MDA含量明显高于对照烟草，igr转基因烟草的光合作用明显低于对照烟草。以上这些指标表明igr转基因烟草对MV处理更为敏感。MV处理条件下igr转基因烟草谷胱甘肽的含量明显高于对照烟草，但是GSH/GSSG的比率明显低于对照烟草，GR酶活性仍明显低于对照烟草，表明在MV胁迫条件下igr转基因烟草中较低的GR酶活性不能有效的将GSSG还原生成GSH。igr转基因烟草中较高的谷胱甘肽净含量说明其谷胱甘肽的合成能力提高，但这仍不能补偿胁迫条件下较低GR酶引起的GSH/GSSG比率降低。MV处理条件下igr转基因烟草和对照烟草相比ASC的含量大大降低，导致DHA/ASC明显升高。测定MDHAR和DHAR的结果表明，MV处理后igr转基因烟草的MDHAR酶活性明显降低，这表明较低的GR酶活性引起ASC再生循环受到抑制。MV处理后较低的GR酶还引起igr转基因烟草中APX的活性大大降低。以上这些结果表明MV处理条件下降低GR酶活性会削弱抗坏血酸－谷胱甘肽循环，从而引起活性氧的大量积累，造成严重的氧化伤害。 4．低温处理的结果和MV处理的结果稍有不同。在GR酶活性较高的i2转基因烟草中所有检测指标与对照烟草无明显差异。而GR酶活性较低的i21、i28和i42植株与对照烟草相比表现出明显差异。低温下生长的对照烟草叶绿素含量明显高于i21、i28和i42植株。i21、i28和i42中活性氧（O2-和H2O2）和MDA的含量都明显高于对照烟草，表明低温处理下i21、i28和i42受到更严重的胁迫伤害。与MV处理后的变化相似，低温处理后i21、i28和i42中较低的 GR酶活性导致GSH/GSSG大大降低，ASC再生循环受抑制，APX活性明显降低，从而使抗坏血酸－谷胱甘肽循环不能高效的清除活性氧，导致ROS和MDA的大量积累，造成严重的低温伤害。

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy

Background. The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. Methods. Seven baboons receiv

A one-year investigation of the relationship between serum GH levels and the growth of F-4 transgenic and non-transgenic common carp Cyprinus carpio

It has been demonstrated that growth hormone (GH) transgenic fish often posses a trait for fast growth. Here, we investigated the growth of F-4 'all-fish' GH transgenic carp Cyprinus carpio and their serum GH levels for a year. The results showed that F-4 all-fish GH transgenic carp were significantly larger in body mass (c. two-fold, P < 0 center dot 001) and body length (c. 1 center dot 3 fold, P < 0 center dot 001), compared with the non-transgenic group. The discrepancy of serum GH levels between the transgenic carp group and control group is 54 fold, when the water temperature was 12-34 degrees C. When the water temperature decreased to 3 center dot 5 degrees C in January, the discrepancy was 256 fold. The serum GH level of the transgenic group was relatively constant, while that of control varied greatly based on month and water temperature. The changes of growth rates between the transgenic group and the control group were similar for a year. Taken together, the results indicated that F-4 all-fish GH transgenic carp had not only higher and constant serum GH levels but also a significant fast-growing effect, compared with the control. To our knowledge, this is the first report on a one-year investigation of growth trait and serum growth hormone level in F-4 all-fish GH transgenic carp.

Elevated ability to compete for limited food resources by 'all-fish' growth hormone transgenic common carp Cyprinus carpio

Food consumption, number of movements and feeding hierarchy of juvenile transgenic common carp Cyprinus carpio and their size-matched non-transgenic conspecifics were measured under conditions of limited food supply. Transgenic fish exhibited 73 center dot 3% more movements as well as a higher feeding order, and consumed 1 center dot 86 times as many food pellets as their non-transgenic counterparts. After the 10 day experiment, transgenic C. carpio had still not realized their higher growth potential, which may be partly explained by the higher frequency of movements of transgenics and the 'sneaky' feeding strategy used by the non-transgenics. The results indicate that these transgenic fish possess an elevated ability to compete for limited food resources, which could be advantageous after an escape into the wild. It may be that other factors in the natural environment (i.e. predation risk and food distribution), however, would offset this advantage. Thus, these results need to be assessed with caution.

Enhanced grass carp reovirus resistance of Mx-transgenic rare minnow (Gobiocypris rarus)

In the interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins have direct antiviral activity and inhibit a wide range of viruses by blocking an early stage of the viral genome replication cycle. However, antiviral activity of piscine Mx remains unclear in vivo. In the present study, an Mx-like gene was cloned, characterized and gene-transferred in rare minnow Gobiocypris rarus, and its antiviral activity was confirmed in vivo. The full length of the rare minnow Mx-like cDNA is 2241 bp in length and encodes a polypeptide of 625 amino acids with an estimated molecular mass of 70.928 kDa and a predicted isoelectric point of 7.33. Analysis of the deduced amino acid sequence indicated that the mature peptide contains an amino-terminal tripartite GTP-binding motif, a dynamin family signature sequence, a GTPase effector domain and two carboxy-terminal leucine zipper motifs, and is the most similar to the crucian carp (Carassius auratus) Mx3 sequence with an identity of 89%. Both P0 and F1 generations of Mx-transgenic rare minnow demonstrated very significantly high survival rate to GCRV infection (P < 0.01). The mRNA expression of Mx gene was consistent with survival rate in F1 generation. The virus yield was also concurrent with survival time using electron microscope technology. Rare minnow has Mx gene(s) of its own but introducing more Mx gene improves their resistance to GCRV. Mx-transgenic rare minnow might contribute to control the GCRV diseases. (C) 2008 Published by Elsevier Ltd.