摘要
油茶是我国特色油料树种,主要栽培分布于南方丘陵酸性红壤地区。茶油因其品质优良而受到国家的高度重视。但是单位面积产量低一直是制约油茶产业快速发展的主要因素。油茶常规育种周期长,常规杂交育种可能伴有遗传累赘。采用分子设计育种新方法改良油茶,可实现单一性状或多个性状的定向改良,大大缩短育种周期,而且不会带有遗传累赘。糖酵解代谢途径是油茶种仁油脂合成的上游代谢途径,其代谢速率直接影响油茶种子油脂合成的效率。果糖1,6-二磷酸醛缩酶不仅是糖酵解代谢途径中的枢纽调控酶,也是油茶种子油脂合成的枢纽调控酶,还可以调控油茶的生殖生长。本文以国审油茶品种‘华硕’种仁为材料,通过构建数字化转录组和表达谱数据库,系统解析了油茶种仁糖酵解途径及其与油茶油脂合成和生殖生长的关系；克隆了4个油茶果糖1,6-二磷酸醛缩酶(CoFBA)家族基因；开展了该酶的亚细胞定位研究；分析了该基因表达与油茶种仁含油率等的关联性；构建了过表达载体和RNA干扰载体,转化野生型拟南芥和油菜,获得了T0代和T1代转基因植株。这些实验结果为油茶分子设计育种技术的开发应用奠定了一定的科学和技术基础。主要研究结果如下：
1.油茶种仁糖酵解途径的分子生物学解析。以果实膨大期(6月)和油脂合成高峰期(10月)的油茶种仁为材料,建立了油茶种仁2个时期的合并转录组和2个不同时期数字化表达谱数据库。数据分析显示,油茶种仁糖酵解途径主要包含306条Unigene,分别归属于23类酶基因,即磷酸葡萄糖变位酶、葡萄糖-6-磷酸酶、葡糖磷酸异构酶、N-乙酰氨基葡萄糖-6-磷酸-异构酶、二磷酸酯酶、磷酸果糖激酶、醛缩酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、二磷酸甘油酸变位酶、醛铁氧还蛋白还原酶、磷酸甘油酸变位酶、烯醇化酶、磷酸烯醇式丙酮酸羧激酶、丙酮酸激酶、丙酮酸脱氢酶(硫辛酰胺)、二氢硫辛酸乙酰转移酶、二氢硫辛酸脱氢酶、丙酮酸脱羧酶、L-乳酸脱氢酶、醛脱氢酶、乙醇脱氢酶、甲醇脱氢酶(细胞色素c)和乙醇脱氢酶(细胞色素c)。这些酶基因大部分为基因家族,其所包含的不同Unigene在油茶种仁发育的不同时期存在差异表达。油茶种仁表达谱糖酵解途径Pathway显著性富集分析结果显示：该代谢途径调控基因表达整体上调,由初期始样本表达量26(6月)上调至高峰期样本表达量336(10月)。根据油茶种仁转录组糖酵解/异生代谢途径Unigene数据分析和不同时期油茶种仁糖酵解代谢功能基因表达差异分析,总结绘制了油茶种仁糖酵解代谢调控途径。在分析过程中发现,转录组数据库中有11条CoFBA Unigene序列,其中有2条在油脂合成高峰期原始表达量是果实膨大期原始表达量的3-4倍,明显高于糖酵解途径中其他调控酶基因的表达量和变化幅度；油茶FBA从甘油代谢途径和脂肪代谢途径直接影响油茶种仁的油脂合成,是影响油茶油脂合成的关键酶之一。
2.油茶FBA家族基因克隆及生物信息学分析。根据转录组分析数据,分别设计特异引物,采用RACE技术,逐一克隆了4个油茶FBA基因的全长cDNA序列,并在NCBI中注册,GenBank登录号分别为JN017093.1、JX914588、JX914589和JX914590。将该4个基因分别命名为CoFBA1、CoFBA2、CoFBA3和CoFBA4。生物信息学方法分析表明,这4个CoFBA基因均无信号肽,为亲水性蛋白,含有典型的1,6-二磷酸果糖醛缩酶活性结构,其中CoFBA1、CoFBA3和CoFBA4存在"VMFEGILLKPS"特异结构域,CoFBA2存在"VLLEGTLLKPN"特异结构域；CoFBA蛋白家族分为A、B两个亚族,CoFBA2归为B亚族,而CoFBA1、CoFBA3和CoFBA4归为A亚族,这意味着分化的先后多少与其活性位点、结构以及功能的差异是密切相关的。空间结构预测CoFBA1和CoFBA4与巴贝斯分枝杆菌1,6-二磷酸醛缩酶3kx6A结构相似,而CoFBA2和CoFBA3分别与兔肌肉醛缩酶31geB和恶性疟原虫二磷酸醛缩酶2pc4C结构相似。
3.油茶FBA蛋白亚细胞定位研究。采用基因枪轰击洋葱表皮细胞法进行亚细胞定位分析,发现CoFBA并不单单定位于细胞质。结果显示,CoFBA1可能定位于细胞核,CoFBA2可能定位于细胞质,CoFBA3可能定位于周质,CoFBA4可能定位于细胞外,这些结果与生物信息学预测的外膜、细胞质、周质和外膜稍有差别。但无论是预测信息还是实验结果,都体现出CoFBA表达部位的多样性,而预测显示这4个醛缩酶均为非分泌性蛋白,如何转运到胞外,可能与其含有的肉豆蔻酰基化及糖基化位点有关。
4.油茶FBA基因的表达对油茶种仁含油率的影响。油茶果实和种仁的表型特征能体现种仁含油率的差异,果实膨大期之前种皮乳白色,种仁白色,此时含油率仅10%左右,果实近成熟期种皮黑褐色,种仁油黄色,此时含油率升至50%左右。采用实时荧光定量PCR分析了8个不同发育时期油茶种仁中CoFBA1、CoFBA2、 CoFBA3和CoFBA4相对表达量,并对照比较了脂肪酸合成关键酶基因CoACP、 CoSAD和CoFAD2相对表达量,结合不同发育时期油茶种仁含油率的变化,采用灰色关联分析法,揭示其综合关联度分别为CoFBA1(0.762817)、CoFBA2(0.649344)、CoFBA3(0.672756)、CoFBA4(0.646704),这与脂肪酸代谢关键酶基因的综合关联度CoACP (0.673442)、oSAD (0.699459)、CoFAD2(0.653404)相比差异不大,说明油茶种仁油脂合成不仅与脂肪酸代谢途径中各关键酶基因相关,而且糖酵解途径中关键酶基因也起到等同甚至更重要的影响。CoFBA基因家族表达调控对油茶种仁含油率具有一定的影响,通过调控CoFBA的表达在一定程度上有可能提高油茶的种仁含油率。
5.超量表达载体和RNA干扰载体构建及遗传转化研究。构建了CoFBA1、 CoFBA1、和CoFBA4的过表达载体,CoFBA家族共抑制RNA干扰Gateway载体和对照用AtFBA家族RNA干扰Gateway载体,农杆菌介导转化哥伦比亚野生型拟南芥,筛选获得了T1代转基因拟南芥；采用Gateway技术构建CoFBA1过表达载体,农杆菌介导转化甘蓝型油菜,筛选获得了T1代‘自交15’转基因甘蓝型油菜,通过表型比较初步证实了CoFBA1促进生殖生长,提高结实率,从而提高产油率的生物学功能。
Camellia oleifera are specialty oil trees in China, which are planted in acidic red soil areas in the South Downs. Camellia oleifera are strongly supported by the state because of high quality tea oil. But the low yield per unit area has been the main factors restricting the rapid development of oil-tea industry. Conventional breeding of Camellia oleifera need long time and may be accompanied by genetic cumbersome. Using a new method of molecular design breeding to modify oil-tea, it not only could be directionally improve the single trait or multiple traits but also could greatly shorten the breeding cycle with out genetic cumbersome. Glycolytic pathway is the upstream metabolic pathway of oil synthesis in Camellia oleifera seeds. The glycolytic metablic rate directly affects the efficiency of fatty acid synthsis in Camellia oleifera seeds. Fructose-1,6-bisphosphate aldolase is the key regulation enzyme in the glycolytic pathway, which not only can regulate plant reproductive growth but can affect fat synthesis. In this paper, state trial oil-tea variety'Huashuo'as material, on the basis of the analysis of the digitized transcriptome and expression profiling database, the full-length cDNA of four key members of Camellia oleifera fructose bisphosphate aldolases(CoFBA) were cloned. The studies of CoFBA subcellular localization were carried out. The correlation between the four CoFBA gene expression and oil content in Camellia oleifera seeds was analyzed. After constructed over-expression and RNA interference vector which were transformed into wild-type Arabidopsis thaliana and Brassica napus, T1transgenic plants were obtained. It laid the scientific foundation for the development of Camellia oleifera molecular design breeding techniques. The main results are as follow:
1. Molecular biology analysis of glycolytic pathway in Camellia oleifera seeds.
Camellia oleifera seeds in fruit enlargement period (June) and oil peak period (October) were chosen as materials. The combined transcriptome of two periods and two different periods digitized expression profiling database were constructed. The data analysis showed that the Camellia oleifera glycolytic pathway contained306unigenes which were attributable to the23genes just as Phosphoglucomutase, Glucose-6-phosphatase, Glucose-6-phosphate isomerase, Fructose-bisphosphatase, N-acylglucosamine-6-phosphate2-epimerase, Phosphofructokinase, Aldolas, Triosephosphate isomerase, Glyceraldehyde-3-phosphate dehydrogenase, Bisphosphoglycerate mutase, Aldehyde ferredoxin oxidoreductase, Phosphoglyceromutase, Enolase, Phosphoenolpyruvate carboxykinase, Pyruvic kinase, Pyruvate dehydrogenase (acetyl-transferring), Dihydrolipoyllysine-residue acetyltransferase, Dihydrolipoic acid dehydrogenase, Pyruvate decarboxylase, L-lactate dehydrogenase, Aldehyde dehydrogenase, Alcohol dehydrogenase, Methanol dehydrogenase (cytochrome c) and alcohol dehydrogenase (cytochrome c). The majority of these genes were gene families, in which unigenes differentially expressed in different developmental period of Camellia oleifera seeds. The glycolytic pathway significant enrichment analysis of camellia oleifera seed expression profiling showed that, the expression abundance of regulating genes in this metabolic pathway increased overall. The initial expression26samples (June) were increased to the peak expression336samples (October). According to the glycolysis/gluconeogenesis metabolic pathway unigene data of Camellia oleifera seeds transcriptome and differential expression of functional genes of Camellia oleifera seeds' glycolytic pathway in different developmental periods, the glycolytic pathway of Camellia oleifera seeds was concluded and drawn. Transcriptome database had11CoFBA unigene sequences, in which the two expression levels in oil synthesis peak period were3-4times than in fruit enlargement period, and significantly higher than the other regulation genes in glycolytic pathway. CoFBA were the key enzymes for oil synthesis of Camellia oleifera to impact from glycerol metabolic pathway to fat metabolic pathway directly.
2. Gene cloning and bioinformatics analysis of CoFBA
According to the data analysis of transcriptome, combined with cloning one by one and RACE, four CoFBA full-length cDNA were cloned and registered in NCBI, GenBank accession number for JN017093.1, JX914588, JX914589and JX914590. The four CoFBA genes were named as CoFBA1、CoFBA2、CoFBA3and CoFBA4. The four CoFBA proteins had no signal peptide and all hydrophilic proteins.They all contained typical FBA activity structure, which CoFBA1, CoFBA2, CoFBA4as 'VMFEGILLKPS' and CoFBA2as 'VLLEGTLLKPN'. The CoFBA family divided into A and B subfamily, in which CoFBA2normalized B subfamily and CoFBA1, CoFBA3, CoFBA4classified as A subfamily. The results suggested what time and how much differentiation were related with active site, structure as well as function differences.The spatial structure predicted that CoFBA1and CoFBA4were similar with Babesia Mycobacterium1,6-bisphosphate aldolase3kx6A structure, while CoFBA2and CoFBA3were closed to rabbit muscle aldehyde reduction the enzyme3lgeB and P. falciparum diphosphate aldolase enzymes2pc4C structure respectively.
3. CoFB A protein subcellular localization
Using gene gun bombardment of onion epidermal cells, CoFBA protein subcellular localization was studied. The results showed that CoFBA were not solely localized in the cytoplasm. CoFBA1may be localized in nucleus, CoFBA2may be localized in cytoplasm, CoFBA3may be localized in periplasm and CoFBA4may be localized in extracellular. But with bioinformatics prediction, CoFBA were located in outer membrane, cytoplasm, periplasm and outer membrane. But whether it was forecast information or was experimental results, it both reflected diversity of CoFBA expression site. Projections indicate that four aldolases were non-secreted protein. How CoFBA to be transported to the extracellular might be related to contain myristylated and glycosylation sites.
4. Influence of CoFBA gene expression on oil content of Camellia oleifera seeds
The differences in oil content could be reflected from Camellia oleifera fruits and seeds phenotype. While the seed coat was milky white and seed kernel white before fruit expansion, oil content was only about10%. Whilee pisperm was dark brown and seed kernel oil yellow, the oil percentage rose to50%. Using real-time quantity PCR technology, the relative expression level in eight different developmental stages of CoFBA1、CoFBA2、CoFBA3and CoFBA4had been analyzed, which contrast with the relative expression level of CoACP, CoSAD and CoFAD2combined with oil content changes, the comprehensive correlation degree had been revealed by gray relational analysis. The comprehensive correlation were CoFBAl (.762817), CoFBA2(0.649344), CoFBA3(0.672756) and CoFBA4(.646704) respectively, which compared insignificant with enzyme of fatty acid metabolism such as CoACP (0.673442), CoSAD (0.699459) and CoFAD2(0.653404). The result showed that Camellia oleifera seeds oil synthesis was not only associated wit the key enzymes in fatty acid metabolism pathway but also the key enzymes in the glycolytic pathway. Because expression and regulation of CoFBA gene family had a certain impact on Camellia oleifera oil content, Camellia oleifera oil content could be improved through genetic improvement means.
5. Constructing and transformation of overexpression vector and RNA interference vector
Overexpression vectors of CoFBA1, CoFBA2, CoFBA3, CoFBA4and RNA interference Gateway vector of CoFBA family were constructed as well as RNA interference Gateway vector of AtFBA family for contrast. The vectors had been transformed to wild Arabidopsis thaliana and the T1transgenic Arabidopsis thaliana had been obtained. Using Gateway technology, the expression vector was constructed and had been transformed to wild Brassica napus. T1transgenic Brassica napus 'inbred15' had been screened. Through the comparison of phenotype, the biological function about promote reproductive growth, improving seeds yield then improving oil production of CoFBA1have been confirmed.
引文
[1]胡芳名,谭晓风,刘惠民.中国主要经济树种栽培与利用[M].中国林业出版社,2006.01.370
[2]陈永忠.油茶栽培技术[M].湖南科学技术出版社,长沙,2008,8：1
[3]陈永忠.油茶优良种质资源[M].中国林业出版社,2008,8：14-15
[4]邓小莲,谢光盛,黄树根.保健茶油的研制及其调节血脂的作用[J].中国油脂,2002,27(5)：96-98.
[5]黄永辉,郑小严,黄红霞等.化妆品用茶油光谱特性分析[J].福建分析测试,2008,17(1)：7-9.
[6]聂海瑜.油茶籽的综合利用[J].粮油加工与食品机械,2004,(6)：39-41
[7]钟旭美,张百刚,朱杰.我国油茶籽的综合利用[J].粮油食品科技,2007,152：34-36.
[8]宋冰蕾,商士,宋湛谦.油茶副产物在绿色表面活性剂中的利用与研究进展[J].生物质化学工程,2010,44(2)：43-47.
[9]谭晓风,漆龙霖,罗春清,贺晶.山茶属植物RAPD分析中琼脂糖凝胶电泳条件的研究[J].中南林学院学报,2000,20(2)：83-85.
[10]谭晓风,漆龙霖,贺晶,桂君,罗春清,胡芳名.山茶属植物油茶组与金花茶组的分子分类[J].中南林学院学报,2005,25(4)：31-34.
[11]陈永忠,张智俊,谭晓风.油茶优良无性系的RAPD分子鉴别[J].中南林学院学报,2005,25(4)：40-45.
[12]邹锋,谭晓风,袁德义,袁军.RAPD分子标记及其在油茶遗传育种中的应用[J].亚热带植物科学,2009,38(1)：70—73.
[13]谭晓风,胡芳名,谢禄山,石明旺,张党权,乌云塔娜..油茶种子EST文库构建及主要表达基因的分析[J].林业科学,2006,42(1)：43-48
[14]石明旺.油茶种子EST文库构建及油脂合成关键酶基因的分离鉴定[D].中南林学院博士学位论文,2004.
[15]Tan Xiaofeng, Chen Hongpeng, Zhang Dangquan, Zhang Lin, Zhang Donglin.SAD and FAD2 cDNA Genes Clones from Camellia oleifera Abel[J]. HortScience,2009,44(4):1088
[16]Wang Baoming, Tan xiaofeng, Zhang Lin, Zhang Donglin.Identification of Two Calmodulin cDNA Genes for Camellia oleifera Abel[J]. HortScience, 2009,44(4):1089
[17]Tan xiaofeng, Wang Baoming, Xie Lushan, Zhang Lin, Zhang Donglin.Cloning Cyclophilin cDNA Gene form Camellia oleifera Abel[J]. HortScience, 2009,44(4):1089
[18]Xiaofeng TAN, Baoming WANG, Lin ZHANG.Cloning and Prokaryotic Expression of a Complementary DNA Gene for Cyclophilin from Camellia Oleifera[J]. Pak.J.Bot.2010,42(6):3847-3855
[19]Bao Meirong, Tan Xiaofeng, Wuyun Tana.Full-length DNA Cloning and Bioinformatic Analysis of Camellia oleifera Seed Ripening-Regulated Protein[J]. CMBB,2010.12
[20]ZHANG Dang-Quan, TAN Xiao-Feng, LIU Qi-Mei, PENG Wan-Xi, ZENG Yan-ling, CHEN Hong-Peng, TIAN Hua..Improved application of Camellia oleifera on biomass energy by enlarging its production. [J].中山大学学报(自然科学版),2007,46(S1)：109-110.
[21]Zhang Dang-quan, Tan xiao-feng, Chen hong-peng.Characteristics and Molecular Genetics of Lipid Biosynthesis in Tea-oil Tree Seed. [J].Seed Science and Biotechnology,2007,1(2)42-47
[22]谭晓风,陈鸿鹏,张党权,曾艳玲,等.油茶FAD2基因全长cDNA的克隆和序列分析[J].林业科学,2008,44(3)：70-75
[23]胡孝义,谭晓风,张党权,曾艳玲,陈鸿鹏,田晓明,刘巧.1个油茶Y2SK2型脱水素的全长cDNA克隆及生理功能分析[J].林业科学,2008,44(10) ：55-62
[24]胡孝义,谭晓风,等.油茶种子水通道蛋白CoPIP1-1的鉴定与分析[J].林业科学,2008,44(12)：48-56
[25]张党权,谭晓风,陈鸿鹏,曾艳玲,等.油茶SAD基因的全长cDNA克隆及生物信息学分析[J].林业科学,2008,44(2)：155-159
[26]李红军,张党权,谭晓风.植物油脂合成途径及酰基载体蛋白基因的特性[J].经济林研究,2007,25(1)：59-65
[27]陈鸿鹏,谭晓风.超氧化物歧化酶(S O D)研究综述[J].经济林研究,2007,25(1)：59-65
[28]张党权,谭晓风,谢禄山,曾艳玲,陈鸿鹏.Sequence Analysis of Key Genes related to unsaturated Fatty Acid Synthesis in the EST library of Camellia Oleifera[J].经济林研究,2007,25(2)：5-8.
[29]张党权,谭晓风,陈鸿鹏.油茶油脂的生物合成及调控基因的特性.[J].中南林业科技大学学报,2007,27(5)：106-111.
[30]谭晓风,张党权,陈鸿鹏等.油茶查尔酮合酶和异构酶基因的cDNA克隆.[J]. 中南林业科技大学学报,2007,27(1)：10-14
[31]胡孝义,谭晓风,田晓明,刘巧,罗茜,谢鹏,曾艳玲.油茶脱水素样蛋白的基因克隆与序列分析及生理功能预测[J].西北植物学报,2008,28(8)：1541-1548
[32]谭晓风,王威浩,刘卓明,张党权,陈鸿鹏,胡芳名.油茶ACP基因的全长cDNA克隆及序列分析[J].中南林业科技大学学报,2008,28(4)：8-14
[33]王保明,谭晓风.植物亲环素基因的结构、功能及表达调控[J].中南林业科技大学学报,2008,28(1)：168-174
[34]田晓明,谭晓风,胡孝义,刘巧,罗茜.油茶苯丙氨酸解氨酶基因的cDNA克隆与序列分析[J].南京林业大学学报(自科版),2009,33(4)：24-28
[35]蒋瑶,谭晓风,张党权,陈鸿鹏.一个油茶金属硫蛋白基因的克隆于序列分析[J].江西农业大学学报,2009,31(4)：699-705
[36]李魏,谭晓风,陈鸿鹏.植物肉桂酰辅酶A还原酶基因的结构功能及应用潜力[J].经济林研究,2009,27(1)：7—12
[37]蒋瑶,谭晓风.植物乙酰辅酶A羧化酶基因的分子生物学研究进展[J].经济林研究,2009,27(2)：111-117
[38]王威浩,谭晓风.植物脂酰—酰基载体蛋白硫脂酶研究综述[J].经济林研究,2009,27(2)：118-124
[39]谭晓风,蒋瑶,王保明,张琳.油茶乙酰辅酶A羧化酶p-CT亚基全长cDNA克隆及序列分析[J].中南林业科技大学,2010,30(2)：1-9
[40]罗茜,谢禄山,谭晓风,田晓明.油茶丙酮酸激酶基因全长cDNA克隆及序列分析[J].中南林业科技大学学报,2010,30(5)：83-90
[41]刘巧,谭晓风,胡孝义,田晓明.油茶14—3—3蛋白基因的cDNA克隆及序列分析[J].江西农业大学学报,2010,32(3)：541-546
[42]胡姣,谭晓风,张琳等.油茶种子总RNA提取及Cod8FAD基因的鉴定[J].浙江林业科技,2010,32(2)：17-21
[43]郭静怡,谭晓风,王威浩,张党权等.油茶FatB基因全长cDNA克隆及序列分析[J].中南林业科技大学学报,2010,30(9)：67-73
[44]仇键.油茶种子油体蛋白基因的分离克隆及其原核表达[D].中南林业科技大学硕士学位论文,2006.
[45]林萍,曹永庆,姚小华,王开良,滕建华.普通油茶种子4个发育时期的转录组分析[J].分子植物育种,2011,9(4)：498-505.
[46]邵奉公.油茶种子油脂合成调控的转录组与表达谱研究[D].中南林业科技大学博士学位论文,2011.
[47]基于油茶组59万条EST序列的转录组学初步分析[J].林业科学,2011,47(2)：161-163.
[48]Trevor J. C. Higgins, David Allsopp, Peter J. Bailey and Eric D. A. D'Souza. The relationship between glycolysis, fatty acid metabolism and membrane integrity in neonatal myocytes[J]. Journal of Molecular and Cellular Cardiology, Volume 13, Issue 6, June 1981, Pages 599-615
[49]秦巧平,张上隆,徐昌杰,己糖激酶与植物生长发育,植物生理学通讯[J].2003,39(1)：1-8
[50]Minet M, Dufour M E, Lacroute F. Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. [J].Plant J., 1992,2:417-422.
[51]Tuckman, D., Donnell, R.J., et al. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegatis. [J].J. Bacteriol. 1997,179:2724-2730.
[52]Kelly GJ, Latzko E. Chloroplast phosphofructokinase. Plant Physiol 1977,60:295.
[53]Rutter W.J..Evolution of Aldolase. Fed. Proc.,1964,23:1248-1257.
[54]OROSZ F, VERTESSY B G, HOLLAN S, et al Triosephosphate isomerase deficiency:predictions and facts[J]. J Theor Biol,1996,182(3):437-447.
[55]HELFERT S, EATEVEZ A M, BAKKER B, et al Roles of triosephosphate isomerase and aerobicmetabolism in Trypanosomabrucei [J]. Biochem J,2001, 357(Ptl):117-125.
[56]周君,徐剑,相入丽,陆小平.蜜蜂磷酸丙糖异构酶基因(AmTpi)全长cDNA的电子克隆及其验证,福建农林大学学报(自然科学版)[J].2009,38(2)：154-159.
[57]卢秉国,何炜毅,申艳红,蒋际谋,陈晓静,陈伟,吕柳新.龙眼子叶胚3-磷酸甘油醛脱氢酶基因的cDNA克隆及序列分析[J].2009,38(5)：507-511.
[58]BRYKSINAV, LAKTIONOV P P. Role ofglyceraldehyde-3-phosphate dehydrogenase in vesicular transport from golgi apparatus to endoplasmic reticulum [J]. Biochemistry (Moscow),2008,73(6):619-625.
[59]HARAM R, SNYDER SH. Nitric oxide-GAPDH-siah:a novel celldeath cascade [J]. Cellular and Molecular Neurobiology,2006,26(4-6):527-538
[60]MARTINEZ P, MARTINW, CERFF R. Structure, evolution and anaerobic regulation of a nuclear gene encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase from maize [J]. JMolBio,l 1989,208(4):551-565.
[61]YANG Y, KWONH B, PENG H P, et a.l Stress responses andmetabolic regulation of glyceraldehyde-3-phosphate dehydrogenase genes in Arabidopsis [J]. PlantPhysio,1 1993,101:(4)209-216.
[62]RUSSELL D A, SACHSMM. Differential expression and sequence analysis of themaize glyceraldehyde-3-phosphate dehydrogenase gene family [J]. PlantCel,l 1989,1(8):793-803.
[63]HAJIREZAEI1M R, BIEMELT S, PEISKERM, et a.l The influence of cytosolic phosphorylating glyceraldehyde-3-phosphate gehydrogenase (GAPC) on potato tubermetabolism [J]. Journal ofExperimentalBotany,2006,57(10):2363-2377.
[64]朱华,李珅,赵瑞强,等.三七植物GAPDH基因克隆及序列分析[J].西北植物学报,2006,26(7)：1316-1319.
[65]岳彩凤,康国章,刘超,等.小麦GAPDH基因克隆及序列分析[J].农业生物技术科学,2008,24(4)：94-98.
[66]ISKANDAR H M, SIMPSON R S, CASU R E, et a.l Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression [J]. PlantMolBiolRep,2004,22(4):325-337.
[67]吴德,吴忠道,余新炳.磷酸甘油酸激酶的研究进展[J].中国热带医学,2005,5(2)：385-387,276.
[68]Colette Valentin,Henrik Birgens,Constantin T1Craescu,A Phosphoglycerate Kinase Mutant (PGK Herlev;D285V) in a Danish Patient With Isolated Chronic Hemolytic Anemia:Mechanism of Mutation and Structure-Function Relationships[J]. HUMAN MUTATION 12:2801287 (1998)
[69]FUJ I.I.,H,KRIETSCH,W.K., YOSHIDA,A.A. single amino acid substitution (Asp leads to Asn) in a phosphoglycerate kinase variant (PGK Munchen) associated with enzyme deficiency [J] J.NIOL.CHEM.255 6421-6423 (1980)
[70]Forsthoefel N R, Cushman M A F, Cushman J C. Postt ranscriptional and Postt ranslational cont rol of enolase epression in the facultative crassulacean acid metabolism plant Messenbry anthemum crystsl l inum[J]. Plant Physiol,1995, 108:1185-1195.
[71]Shailesh KL, Chwenfang L, Martin M. Differential regulation of enolase during anaerobiosis in maize[J]. Plant Physiol,1998,118:1285-1293.
[72]Grodzinski B.,Jiao J.R, Knowles V.L,et al.Photosynthesis and carbon partitioning in transgenic tobacco plants deficient in leaf cytosolic pyruvate kinase [J]. Plant Physiology,1999,120:887-895.
[73]Hems D A,Raht E A,Verrinder T R.Fatty acid synthesis in liver and adipose tissue of normal and genetically obese (ob/ob) mice during the 24-hour cycle[J]. Biochem J,1975,150(2):167-173.
[74]Duff SM G,Lefebvred D,Plaxtonw C.Purification and characterisation of a phosphoenol pyruvate phosphatase from Brassica nigra suspension cells [J].Plant Physiol,1989,90:734-741.
[75]Duff SM G,Moorhead GB G,Lefebvred D et al.Phosphate starvation inducible bypasses of adenylate and phosphate dependent glycolytic enzymes in Brassica nigra suspensin cells[S].Plant Physiol,1989,90:1275-1278.
[76]Sung S S,Dian-Peng X,Galloway C M et al.A reassessment of glycolysis and gluconeogenesis in higher plants[J].Physiol Plant,1987,72:650-654.
[77]Theodorou M E,Elrifi IR,Turpin D H et al.Effects of phosphorus limitation on respiratory me tabolism in the green alga selenstrum minutum[J].Plant Physiol,1991,95:1089-1095.
[78]Gottlob-Mchugh S QSangwan R S,Blakley S D et al.Normal growth of transgenic tobacco plants in the absence of cytosolic pyruvate kinase[J].Plant Physiol,1992,100:820-825.
[79]S Cortassa, M A Aon, A A Iglesias, D Lioyd.代谢与细胞工程导论(影印版)[M].科学出版社,北京,2004,9.
[80]Schnarrenberger C., Jacobshagen S., Muller B. and Kruger I.. Evolution of isozymes of sugar phosphate metabolism in green algae. Prog. Clin. Biol. Res., 1990,344:743-764.
[81]Von der Osten C.H.,Barbas C.F.,Wong C.H.and Sinskey A.J..Molecular cloning, nucleotide sequence and fine-structural analysis of the Corynebacterium glutamicum fda gene:structural comparison of C.glutamicum fructose-1, 6-biphosphate aldolase to class I and class II aldolases.Mool. Microbiol, 1989,3:1625-1637
[82]Lebherzh G.and Rutter W.J..Distribut ion of fructose diphosphate aldolas e variants in biological systems.Bioch.emistry,1969,8(1):109-121
[83]Lebherz H.G.,Leadbetter M.M.and Bradshaw R.A..Isolation and characterization of the cytosolic and chloroplast forms of spinach leaf fructose diphosphate aldolase J.Biol.Chem.,1984,259:1011-1017
[84]Pelzer-Reith B.,Penger A.and Schnarrenberger C.Plant aldolase:cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach.Plant Mol. Biol,1993,21:331-340
[85]Tsutsumi K.,Kagaya Y.,Hidaka S.,Suzuki J.,Tokairin Y.,Hirai T.,Hu D.L.Ishikawa K. and Ejiri S..Structural analysis of the chloroplastic and cytoplasmic aldolase-encoding genes implicated the occurrence of multiple loci in rice.Gene,1994,141:215-220
[86]吴耀荣,谢旗.ABA与植物胁迫抗性.植物学通报,2006,23：511-518
[87]Rajjou L.,Belghazi M.,Huguet R.,Robin C.,Moreau A.,Job C.and Job D. Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms.Plant PhysioL,2006, 141:910-923.
[88]Konishi H.,Yamane H.,Maeshima M.and Komatsu S..Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling.Plant Mol.Biol.,2004,56:839-848
[89]Osakabe Y.,Maruyama K.,Seki M.,Satou M.,Shinozaki K.and Yamaguchi-Shinozaki K..Leucine-rich repeat receptor-like kinasel is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis.Plant Cell,2005,17: 1105-1119
[90]Jiang Y.,Yang B.,Harris N.S.and Deyholos M.K..Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots.J.Exp.Bot,2007, 58:3591-3607
[91]张其德.盐胁迫对植物及其光合作用的影响[J].植物杂志,1999,6：32-33.
[92]Blumwald E.. Sodium transport and salt tolerance in plants. Current Opinion in Cell Biology,2000,12:431-434.
[93]Haake V, Geiger M, Walch-Liu P, et al. Changes in aldolase activity in wild-type potato plants are important for acclimation to growth irradiance and carbon dioxide concentration, because plastid aldolase exerts control over the ambient rate of photosynthesis across a range of growth conditions. Plant J,1999, 17:479-489.
[94]Yamada S.,Komori T.,Hashimoto A.,Kuwata S.,Imaseki H.and Kubo T..Differential expression of plastidic aldolase genes in Nicotiana plants under salt stress.Plan Sci.,2000,154:61-69
[95]Jiang Y., Yang B., Harris N.S. and Deyholos M. K.. Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. J.Exp. Bot, 2007,58:3591-3607.
[96]Ndimba B.K., Chivasa S., Simon W.J. and Slabas A. R.. Identification of Arabidopsis salt and osmotic stress responsive proteins using two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics,2005,5:4185-4196.
[97]Sarry J.E.,Kuhn L.,Ducruix C.,Lafaye A.Junot C.,Hugouvieux V.,Jourdain A.,Bastien O.,Fievet J.B.,Vailhen D.,Amekraz B.,Moulin C.,Ezan E.,Garin J.and Bourguignon J. The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses.Proteomics, 2006,6:2180-2198
[98]Provart N.J.,Gil P.,Chen W.,Han B.,Chang H.S.,Wang X.and Zhu T..Gene expressionphenotypes of Arabidopsis associated with sensitivity to low temperatures.Plant Physiol.,2003,132:893-906
[99]路玮.拟南芥果糖1,6-二磷酸醛缩酶家族分析[D].泰安：山东农业大学,2011.
[100]罗佳,周建平,谭惠元.红花油茶的主要成分分析[J].现代食品科技,2010,26(1)：109-113.
[101]J.里斯梅尔,R.特里斯维,L.威尔米特尔.糖酵解速率增加的转基因植物细胞核植物[P].中华人民共和国专利局,1998.
[102]Csaba Cseke and Bob B.. An enzyme synthesizing fructose 2,6-bisphosphate occurs in leaves and is regulated by metabolite effectors[J].FEBS LETTERS, 1983,155(1):139-142.
[103]Emile Van Schaftingen and Henri-Gery Hers, Fructose 2,6-bisphosphate and the control of the energy charge in higher plants, Emmanuel Mertens[J].FEB 05063, 1987,221(1):124-128.
[104]Brendan Leighton, Rui Curi and Anwar Hussein et al. Maximum activities of some key enzymes of glycolysis, glutaminolysis, Krebs cycle and fatty acid utilization in bovie pulmonary endothelial cells, [J].FEB 05350,1987, 225(1,2):93-96.
[105]Emmanuel Mertens.Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme, [J].FEBS 09857,1991,285(1):1-5.
[106]Greg B.G.. Moorhead and William C.Plaxton. Evidence for an interaction between cytosolic aldolase and the ATP-and pyrophosphate-dependent phosphofructokinases in carrot storage roots, [J]. FEBS 11799,313(3):277-280.
[107]Federico Valverde, Manuel Losada, Aurelio Serrano. Engineering a central metabolic pathway:glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene[J].FEBS Letters 1999, 449:153-158.
[108]Valentine B. Andelaa,b, Edward M. Schwarza, Regis J. O A. genome-wide expression profile and system-level integration of nuclear factor kappa B regulated genes reveals fundamental metabolic adaptations during cell growth and survival[J]. FEBS Letters2005,579:6814-6820.
[109]Galina Polekhina, Abhilasha Gupta and Bryce J.W. van Denderen er al. Structural Basis for Glycogen Recognition by AMP-Activated Protein Kinase[J]. Structure,2005,13,1453-1462.
[110]Sudesh Kumar Yadavl, Sneh L. Singla-Pareek and M.K. Reddy et al.Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress[J]. FEBS 30088,2005,579:6265-6271.
[111]Jinpeng Zhang, Tingsong Liu, Junjie Fu et al. Construction and application of EST library from Setaria italica in response to dehydration stress [J].Genomics 2007,90:121-131.
[112]Oliver Bartsch, Martin Hagemann, Hermann Bauwe. Only plant-type (GLYK) glycerate kinases produce D-glycerate 3-phosphate[J]. FEBS Letters 2008,582:3025-3028.
[113]Rusk N.Cheap third-generation sequencing [J]. Nature,2009,6(4):244-245.
[114]Ouyang Z,Zhou Q,Wong WH.ChIP-Seq of transcription factors predicts absolute and differential gene expression in embryonic stem cells[J]. Proc Natl Acad Sci USA,2009,106(51):21521-21526.
[115]Morin RD,O'Connor MD,Griffith M,et al.Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. [J]. Genome Res,2008,18(4):610-621.
[116]Hou B K, Zhou Y H, Wang L H, et al. Chloroplast transformation in oilseed rape[J]. Transgenic Research,2003,12:111-114.
[117]Sugiura M, Hirose T, Sugita M. Evolution and mechanism of translation in chloroplasts[J]. Annu. Rev. Genet,1998,32:437-459
[118]王曦,汪小我,王立坤等.新一代高通量RNA测序数据的处理与分析[J].生物化学与生物物理进展,2010,(8)：43-46.
[119]Mortazavi A., Williams B.A., McCue K., Schaeffer L., and Wold B.,2008, Mapping and quantifying mammalian transcriptomes by RNA-Seq [J]. Nature, 5(7):621-628
[120]Marioni J.C., Mason C.E., Mane S.M., Stephens M., and Gilad Y.RNA-seq:An assessment of technical reproducibility and comparison with gene expression arrays [J]. Genome Research,2008,18(9):1509-1517
[121]Hoen P.A.C., Ariyurek Y, Thygesen H.H., Vreugdenhil E., Vossen R.H.A.M., Menezes R.X., Boer J.M., Ommen G.J.B., and Dunnen J.T.Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms, Nucleic Acids Research [J].2008,36(21):e141
[122]Tang F., Barbacioru C., Wang Y.Z., Nordman E., Lee C., Xu N. L., Wang X.H., Bodeau J., Tuch B.B., Siddiqui A., Lao K.Q., and Surani M.A., mRNA-Seq whole-transcriptome analysis of a single cell [J]. Nature,2009,6(5):377-382.
[123]Wilhelm B.T., and Landry J.R..RNA-Seq-quantitative measurement of expression through massively parallel RNA-sequencing[J].Methods,2009,48(3): 249-257
[124]Nagalakshmi U., Wang Z., Waern K., Shou C., Raha D., Gerstein M., and Snyder M.The transcriptional landscape of the yeast genome defined by RNA sequencing [J].2008, Science,320 (5881):1344-1349.
[125]Yassoura M., Kaplana T., Fraserb H.B., Levinb J.Z., Pfiffnerb J., Adiconisb X., Schrothd G., Luod S., Khrebtukovad I., Gnirkeb A., Nusbaumb C., Thompsonb D.A., Friedmana N., and Regevb A..Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing [J]. PNAS,2009,106(9): 3264-3269.
[126]Sultan M., Schulz M.H., Richard H., Magen A., Klingenhoff A., Scherf M., Seifert M., Borodina T., Soldatov A., Parkhomchuk D., Schmidt D., O'Keeffe S., Haa S.s, Vingron M., Lehrach H., and Yaspo M.L..A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome [J].Science,2008,321(5891):956-960.
[127]Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M. Blast2GO:a universal tool for annotation, visualization and analysis in functional genomics research [J]. Bioinformatics,2005,21 (18):3674-3676.
[128]Morrissy, A. S., R. D. Morin, et al. Next-generation tag sequencing for cancer gene expression profiling. [J]. Genome Res.2009,19(10):1825-1835.
[129]Audic, S. and J. M. Claverie. The significance of digital gene expression profiles. [J]. Genome Res,1997,7(10):986-95.
[130]Benjamini, Y and D. Yekutieli (2001). "The control of the false discovery rate in multiple testing under dependency." The Annals of Statistics 29:1165-1188.
[131]Kanehisa, M., M. Araki, et al. (2008). "KEGG for linking genomes to life and the environment." Nucleic Acids Res 36(Database issue):D480-4.
[132]尚丹,卢铭.生物信息学概述[J].北京大学学报,2001,33(1)：92-95.
[133]Brunak S., von Heijne G, and Nielsen H. SignalP 4.0:discriminating signal peptides from transmembrane regions Petersen TN [J].Nature Methods,2011, 8:785-786.
[134]周蕾,顾建,N-糖基化位点鉴定方法和非经典N-糖基化序列,生命科学,2011,23(6)：605-611.
[135]Lazikani B. Protein structure prediction. Curr Opin Chem Biol 2003; 5:51-56.
[136]Benkert P, Biasini M, Schwede T. (2011). "Toward the estimation of the absolute quality of individual protein structure models." Bioinformatics,27(3):343-50.
[137]Andreas D. Baxevanis, B. F. Francis Ouellette.生物信息学一基因和蛋白质的实用指南[M].清华大学出版社,北京,2000,246-250.
[138]杨朝辉,雷建军.绿色荧光蛋白基因研究进展[J].生物学杂志,2000,5(17)：12-14.
[139]周波.DHHC型锌指蛋白基因OsDHHC1在水稻株型构建中的功能分析[D].湖南大学博士学位论文,2011.
[140]Francesco Macri and Angelo Vianello. ADP-or pyrophosphate-dependent proton pumping of pea stem tonoplast-enriched vesicles[J]. FEB 04593,1987, 215(1):47-52.
[141]周善跃.三个烟草品种瞬时表达OsWAK1::GFP的差异.中国农学通报,2009,25(8)：58-61.
[142]夏平安,刘维全,江禹,孙绍光,杨淑艳,王吉贵.家蝇卵黄蛋白基因启动子区的克隆与活性分析.遗传学报,2004,31(7)：688-694.
[143]Chalfie M, Tu Y, Euskirchen G, Ward W W, Prasher D C. Green fluorescent protein as a marker for gene expression. Science,1994,263:802-805.)
[144](Zhou S Y, Zhao W S, Li H, Guo Z J, Peng Y L. Characterization of a novel RING finger gene OsRFP1, which is induced by ethylene, salicylic acid and blast fungus infection in rice. Phytopathology,2008,156:396-402.//
[145]Lin R M, Zhao W S, Meng X B, Wang M, Peng Y L. Rice gene OsNAC19 encodes a novel NAC domain transcription factor and responds to infection of Magnaporthe grisea. Plant Science,2007,172:120-130.)
[146]刘中成,赵锦,刘孟军.：mRNA差异显示技术评述[J].分子植物育种, 2004,2(6)：895-900.
[147]Gong W, Shen YP, Ma LG. Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes [J]. Plant Physiol,2004,135:773-782.
[148]Shimkets RA, Lowe DG, Tai JT,et al. Gene expression analysis by transcript profiling coupled to a gene database query [J]. Nat Biotechnol.1999,17: 798-03
[149]邓文星,张映.实时荧光定量PCR技术综述[J].生物技术通报,2007,5：93-95,103.
[150]谭晓风,袁德义,袁军,邹锋,谢鹏,苏勇,杨定桃,彭建桃.大果油茶良种‘华硕’[J].林业科学,2011,47(12)：184.
[151]王保明.油茶ACCase基因的克隆及功能研究[D].中南林业科技大学,博士学位论文,2012.
[152]谢鹏.38个油茶优良无性系综合评价研究[D].中南林业科技大学硕士学位论文,2010.
[153]谭晓风,王威浩,刘卓明,张党权,陈鸿鹏,胡芳名,油茶ACP基因的全长cDNA克隆及序列分析[J].中南林业科技大学学报,2008,28(4)：8-14.
[154]张党权,谭晓风,陈鸿鹏,曾艳玲,蒋瑶,李魏,胡芳名.油茶SAD基因的全长cDNA克隆及生物信息学分析[J].林业科学,2008,44(2)：155-159.
[155]谭晓风,陈鸿鹏,张党权,曾艳玲,李魏,蒋瑶,谢禄山,胡孝义,胡芳名.油茶FAD2基因全长cDNA的克隆和序列分析[J].林业科学,2008,44(3)：70-75.
[156]许亮,卢向阳,田云.后基因组时代基因功能分析的策略[J].中国生 物工程杂志,2003,23(8)：29-34.
[157]胡孝义.油茶种子活性氧相关基因的克隆及树体RNA干扰技术研究[D].中南林业科技大学博士学位论文,2009.
[158]Clough S.J., Bent A. Floral dip:A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. [J]. Plant J.1998,16:735-743.