【作者】 刘颖丽；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 生态学， 2008， 博士

【摘要】 光合作用是地球上最重要的化学反应,光能的捕获和传递过程将会直接影响整个生物体的光合作用表现。在高等植物中,光系统II捕光色素蛋白复合体LHC在光合作用过程中发挥着极其重要的作用。研究表明LHCII主要的功能有以下四个方面:捕获和传递光能、光保护和过剩能量耗散、调节光能在两个光系统中分配和维持类囊体膜的结构等。光合作用反应植物对外界物质的同化能力,是植物的重要生理活动指标,在一定程度上对植物的生长速度起着决定性的作用。竹子具有生长快、产量高等特点,意味着竹子具有较强的同化能力,本研究从竹子捕光色素结合蛋白基因的分离入手开展了工作,主要结论如下:1.通过RACE、RT-PCR等方法从毛竹克隆到了6个捕光色素结合蛋白基因cab-PhE3、cab-PhE1、cab-PhE5、cab-PhE8、cab-PhE10、cab-PhE11,分别属于光系统Ⅱ的lhcb1、lhcb2、lhcb3、lhcb4、lhcb5、lhcb6类基因家族。通过生物软件分析,该6个基因的CDS编码区均约在800~900 bp左右,其编码蛋白的分子量在28~30kD之间;与国际核酸和蛋白质数据库联网分析得知,6个基因与其它物种中的同类基因均有较高的相似性,其相似性多数在80%以上,特别是与水稻、玉米、大麦等亲缘关系较近的单子叶植物中该类基因的相似性更高,多数在90%以上;蛋白功能预测分析表明,该6个基因均含有典型的叶绿素a/b结合位点,推测其功能可能是与色素分子结合。对6个基因编码的蛋白质进行比较发现:Cab-PhE3与Cab-PhE1和Cab-PhE5之间的相似性分别为72.6%和65.7%,而与Cab-PhE8、Cab-PhE10、Cab-PhE11之间的相似性分别为29.4%、41.4%和24.8%。Cab-PhE1和Cab-PhE5之间的相似性为66.5%。毛竹捕光色素结合蛋白基因的分离,丰富了该基因家族成员,为进一步了解lhcb家族基因在不同植物尤其是在竹类植物中的结构、功能及表达情况奠定了基础。2.实现了毛竹cab-PhE3、cab-PhE1、cab-PhE5基因在大肠杆菌BI21(DE3)中高效表达。通过添加酶切位点的方法将cab-PhE3、cab-PhE1、cab-PhE5基因编码成熟蛋白的序列部分插入到原核表达载体pET-23a的相应位点,构建成原核表达载体pET- cab-PhE3 -mature、pET- cab-PhE1-mature和pET- cab-PhE5-mature。经IPTG诱导后,蛋白电泳结果显示重组质粒在大肠杆菌中能够高效表达,其诱导表达蛋白的分子量均与cab-PhE3、cab-PhE1、cab-PhE5基因编码的成熟蛋白分子量相接近,这为进一步研究捕光叶绿素a/b结合蛋白与色素的体外重组特性奠定基础。3.研究了毛竹cab-PhE3、cab-PhE1、cab-PhE5、cab-PhE8、cab-PhE10、cab-PhE11基因的时空表达。毛竹叶绿素荧光动力学研究结果显示,不同时间强光照射处理的Fv/Fm、qP、Yield、ETR值均随着光照时间的延长而降低,而F0、qN则随光照时间的延长而成上升趋势。实时定量PCR研究表明,cab-PhE3、cab-PhE1、cab-PhE5、cab-PhE8、cab-PhE10、cab-PhE11基因在不同光处理条件下表达明显不同。分别对强光照射2 hr、4 hr、6 hr的毛竹植株的lhcb基因的表达水平进行研究,发现cab-PhE3、cab-PhE1、cab-PhE5、cab-PhE8、cab-PhE10、cab-PhE11基因表达量呈现规律性变化,6个基因均随光照时间的延长而表达量减少。其中cab-PhE3和cab-PhE1基因表达情况变化较为明显。由此表明,毛竹捕光色素结合蛋白基因cab-PhE3、cab-PhE1、cab-PhE5、cab-PhE8、cab-PhE10、cab-PhE11在强光抑制的光保护过程中发挥着一定作用。更多还原

【Abstract】 Photosynthesis, occured at the thylakoid in chloroplast, as the most important biochemical reaction in the world, affects directly the photosynthetic process by absorbing and transferring solar energy. The peripheral light-harvesting complex LHC of photosystemⅡ(LHCⅡ) plays a significant role in photosynthesis of higher plants. In addition to light-harvesting, photoprotection via heat dissipation, energy modulation and distribution, and maintenance of thylakoid are all the main functions of LHCⅡ. Photosynthesis, as the most physiological indicator, plays a decisive role in plant growth to some extent, which representing the assimilation capacity of plant.Bamboo has great light assimilation capacity for fast growth and abundant output. It is significant to focus on the molecular mechanism of bamboo photosynthesis through bio-technology, and valuable for the development and utilization of bamboo resource. Isolation and expression of the light-harvesting chlorophyll a/b binding protein genes of bamboo were performed in this paper. The major results were described as follows:1. light-harvesting chlorophyll a/b binding protein genes (cab-PhE3, cab-PhE1, cab-PhE5, cab-PhE8, cab-PhE10 and cab-PhE11) ,which belong to lhcb1, lhcb2, lhcb3, lhcb4, lhcb5 and lhcb6 gene family respectively, were isolated from Moso (Ph. edulis) through RT-PCR and RACE technology.Results showed that, the CDS of 6 genes were all about 800~900 bp, and molecular weight of the deduced proteins were all between 28~30kD by bioinformatics analysis. According to International nucleic acid and protein databases, the deduced proteins were highly homologous to other CAB proteins, most of them more than 80%;especially to the monocotyledon plants, such as rice, maize, barely and so on, more than 90% respectively. The motifscan analysis indicated that, these proteins all had chlorophyll a/b binding domain which may bonding chlorophyll. The similar coefficient among Cab-PhE3 and Cab-PhE1, Cab-PhE5 were 72.6%, 65.7% respectively; and that of Cab-PhE8, Cab-PhE10, Cab-PhE11were 29.4%, 41.4% and 24.8% respectively; that between Cab-PhE1 and Cab-PhE5 was 66.5%. Isolation of the six light-harvesting chlorophyll a/b binding protein genes of LHCⅡfrom bamboo is very helpful to the further research on the structure and function of LHCⅡin different plants. 2. The light harvesting chlorophyll a/b-binding protein genes (cab-PhE3, cab-PhE1 and cab-PhE5) of Phyllostachys edulis were highly expressed in Escherichia coli.The prokaryoti expression vector, pET- cab-PhE3 -mature, pET- cab-PhE1 -mature and pET- cab-PhE5-mature,were constructed by adding restriction enzyme sites to the sequence encoding mature protein of cab-PhE3, cab-PhE1 and cab-PhE5 and then ligating to the multiple clone sites of pET-23a. The protein electrophoresis revealed that mature proteins of cab-PhE3, cab-PhE1 and cab-PhE5 were highly expressed in E. coli induced by IPTG. Molecular weight of the inducible proteins were all approximate to that of the mature protein, which would be very important for further research on in vitro reconstitution of light-harvesting Chl a/b complexes.3. The space-time expression of cab-PhE3, cab-PhE1, cab-PhE5, cab-PhE8, cab-PhE10 and cab-PhE11 genes from Ph. edulis was studied.Chlorophyll fluorescence parameters of Ph. edulis were studied in this paper. The plant was illuminated by different time of strong light treatment (2 hr, 4 hr and 6 hr). All chlorophyll fluorescence parameters were changed: Fv/Fm, qP, Yield and ETR were decreasing, while Fo and qN were increasing with the time of strong light. The expression lever variance of cab-PhE3, cab-PhE1, cab-PhE5, cab-PhE8, cab-PhE10 and cab-PhE11 genes was displayed by Real time PCR. The expression level of the six genes all decreased, especially cab-PhE3 and cab-PhE1 changed obviously. It could be deduced that cab-PhE3, cab-PhE1, cab-PhE5, cab-PhE8, cab-PhE10 and cab-PhE11 genes may be related with photoinhibition.更多还原