西藏砂生槐水土保持效益分析

通过对雅江下游砂生槐水土保持功能进行的试验研究,结果表明:砂生槐的枝叶及枯落物可截留和吸收大量降雨,减小雨水对土壤的侵蚀,植被覆盖度越大,效果越明显;枯落物及根系的活动明显地改善土壤理化学性质,降低土壤容重,增加土壤孔隙度,提高土壤保水和透气能力等。砂生槐枝叶茂盛,根系发达,可作为保水保土、退耕还林还草的树种大面积推广。     

拉萨半干旱河谷砂生槐灌丛生态恢复过程的群落特征与土壤微生物动态分析

为揭示拉萨半干旱河谷地带性植被砂生槐灌丛生态恢复过程的特征动态,采用空间序列代替时间序列研究方法,将处于不同封育年限(3,6,9年及CK)的砂生槐植被作为植被恢复的典型群落,对其高度、盖度、密度、生物量、多样性指数、种群生态位宽度及土壤细菌、放线菌、真菌等微生物动态进行分析.结果表明:1)采取封育措施后,外界干扰减少,从而增加物种多样性和植物特征值,使生产力得到提升;2)砂生槐生态位宽度最大,是河谷地带群落中的建群种及优势种;藏沙蒿分布较为均匀,是一种泛化种;3)群落中土壤微生物的数量与封育时间呈正比,随着封育时间的延长而越发丰富,说明在砂生槐群落封育恢复过程中,土壤环境有较为明显的改善.     

柳树SjMIPS基因的克隆及其表达分析

肌醇磷酸合成酶(MIPS)以葡萄糖-6-磷酸为合成前体,是肌醇合成过程中关键酶。MIPS生成的肌醇在耐盐植物的耐盐机制中起到渗透压调节物的作用。该研究从柳树(Salix Jiangsuensis 2345)叶片中成功克隆出MIPS基因,命名为SjMIPS。对扩增的基因进行生物信息学分析显示柳树SjMIPS基因包含1 533 bp的开放阅读框,编码1个511个氨基酸的蛋白质。蛋白质结构域分析显示SjMIPS基因含有4个保守的结构域,结构域1(GWGGNNG),结构域2(LWTANTERY),结构域3(NGSPQNTFVPG)和结构域4(SYNHLGNNDG),属于典型的MIPS基因。系统进化树分析表明柳树SjMIPS基因与杨树Pt MIPS基因亲缘关系较近。实时荧光定量PCR结果显示柳树SjMIPS基因在171 m M Na Cl盐胁迫处理24—48 h后上调,表明柳树MIPS基因是盐胁迫诱导型基因。该研究表明柳树SjMIPS基因为盐胁迫应答基因,在参与肌醇的生物合成、调节渗透压方面起着重要作用。     

Cloning and sequence analysis of nine novel MYB genes in Taxodiaceae plants

Myeloblastosis(MYB) is one of the largest transcribed factor families in plants. To gain an overall picture of the evolution of MYB genes in relict plants, we cloned nine novel MYB genes in Taxodiaceae plants(Taxodium distichum, Taxodium ascendens, Cryptomeria japonica var. Sinensis, Cryptomeria japonica cv. Araucarioides, Cryptomer Japonica, Metasequoia glyptostroboides, Cunninghamia lanceolata, Taiwania cryptomerioides and Glyptostrobus pensilis). The deduced amino acid sequences for MYBs showed that the nine MYB proteins contained two DNA binding domains. The first domain is from amino acid position 29 to 78, wherein three tryptophanes at 33, 53 and 73 were separated by 19 amino acids, respectively. The second domain is from amino acid position 82 to 127, wherein three tryptophanes at 86, 105 and 124 were separated by 18 amino acids, respectively, whereas the first tryptophane at amino acid position 86 is replaced by a phenylalanine. The characterization of these conserved domains at nine MYBs indicated that they all belong to the R2R3-MYB group. The secondary structure analysis showed that α-helix and β-turn are the major motifs of the predicted secondary structure of MYBs. The three dimensional model of each MYB protein showed that the structure is like clip, making it more flexible and mobile. The similarities between the nine MYB proteins in Taxodiaceae were calculated. The highest identical value of 99% is between CjsMYB, CjMYB and CjaMYB, whereas the lowest value of 82% is between TaMYB and ClMYB. According to the phylogenetic tree, the distances between different genera were relatively large whereas those within genera were relatively small. As expected, accessions of the same genus formed a subgroup before being grouped with other genera.     

2个野扁桃自交不亲和SFB基因全长的克隆与序列分析

为给野扁桃的遗传改良和自交不亲和机制的进一步研究提供理论依据,以新疆野扁桃花药为试材,利用RT-PCR和RACE技术克隆野扁桃自交不亲和性花粉特异性决定因子编码基因SFB全长序列。采用BLAST进行序列比对,ORF Finder寻找开放阅读框,CDD分析蛋白保守结构域,Prot Param分析蛋白理化性质,TMHMM预测蛋白跨膜区,Signal P预测蛋白信号肽,Sub Loc预测蛋白亚细胞定位,SOPMA分析蛋白二级结构,DANMAN进行氨基酸多序列比对,MEGA6进行系统进化分析,Prot Fun预测蛋白功能。结果表明:克隆到的Pt SFB16基因和Pt SFB17基因属于F-box基因家族,与其它多种植物的SLF/SFB基因的序列相似度为88%,推导的氨基酸序列均具有F-box蛋白典型结构;Pt SFB16基因ORF长1 146 bp,编码381个氨基酸,Pt SFB17基因ORF长1 131 bp,编码376个氨基酸;预测2个SFB蛋白均为略显亲水性、不稳定的细胞质蛋白,二级结构均以α-螺旋,延伸链和无规则卷曲为主,SFB/SLF蛋白在蔷薇科李属植物中具有较高的系统进化一致性,种间同源性可能大于种内同源性,SFB蛋白可能的功能主要有辅助因子生物合成、能量代谢和裂合酶。     

美洲黑杨木质素合成关键基因的克隆及反义表达载体的构建

以美洲黑杨Populus deltoides新萌叶片为材料,通过自行设计引物,用RT—PCR的方法克隆了美洲黑杨木质素合成关键基因4CL和CAD基因部分序列。测序结果表明:这两个基因片段长度分别为859 bp和493 bp。此外,人工合成了4CL基因木质部特异表达启动子4CLp,长度为1 180 bp。分别用限制性内切酶Hind III/BamH I、BamHI/Sma I和Sma I/Sac I对4CLp、4CL和CAD基因序列进行双酶切,同时用Hind III/Sac I对pBI121表达载体进行双酶切,回收了目的片段。酶切回收后用T4DNA连接酶克隆到植物表达载体pBI121中,并转化至大肠杆菌感受态细胞DH5α。经质粒PCR和双酶切分析,确定获得了pBI—4CLp—a4CL—aCAD反义植物表达载体。     

球毛壳菌(Chaetomium globosum)小热休克蛋白HSP22.4基因克隆及表达

小热休克蛋白(sHsps_)是生物体中参与防御反应的内源细胞保护因子。为了研究球毛壳菌中(Chaetomiumglobosum)中的小热休克蛋白(AY491980)。对球毛壳菌的小热休克蛋白进行了克隆和在大肠杆菌中进行了表达。BlastX分析表明球色壳菌的小热休克蛋白与粗糙脉孢菌(Neurosporacrassa)小热休克蛋白在氨基酸上的序列同源性最高,为65%。将HSP22.4基因编码区插入原核表达载体pGEX-4T-2、构建成表达质粒pGEX-HSP。利用pGEX-HSP质粒转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在50kD处有一特异性融合蛋白带,与预期相符,说明HSP22.4基因已经在大肠杆菌中表达。本研究为进一步研究小热休克蛋白的功能奠定了基础。图6参15。     

Cloning and expression analysis of the FvNCED3 gene and its promoter from ash(Fraxinus velutina)

The 9-cis-epoxycarotenoid dioxygenase(NCED)gene is rate-limiting in abscisic acid(ABA) biosynthesis.In this study, an NCED gene, designated FvNCED3(KY008746), was cloned from velvet ash(Fraxinus velutina Torr.) with a RACE method. The full length c DNA of FvNCED3 encodes a 573-amino acid polypeptide.Sequencing analysis showed that the FvNCED3 protein was highly homologous to other NCED proteins. The expression patterns of FvNCED3 in different ash organs were analyzed by real-time PCR which revealed that FvNCED3 expression levels were highest in leaves and lowest in roots. The gene expression patterns of FvNCED3 under abiotic stress indicated that its expression increased under drought, salt and ABA stress and decreased due to high and low temperatures. There were no obvious changes under ultraviolet light. The 1094-bp upstream sequence 5' flank regulation region of the FvNCED3 gene was also cloned from ash using the Genome Walking method. To assess the activity of the FvNCED3 promoter, a p FvNCED3 p::GUS plant expression vector was constructed for tobacco transformation. GUS expression of the FvNCED3 GUS enzyme activity was detected in almost all transgenic tobacco tissues, especially in the young leaves,stigma, anther, ovule and ovary. After treating the transgenic tobacco with NaCl and placing it under drought stress, GUS staining of tobacco leaves increased compared with that under normal growth conditions. This result indicates that gene expression driven by the FvNCED3 promoter can be induced by salt and drought stress.     

多穗柯4-香豆酸辅酶A连接酶基因的克隆和表达分析

为了克隆多穗柯4-香豆酸辅酶A连接酶(4-coumarate-CoAligase,4CL)基因,了解其表达情况,从而为深入研究4CL基因分子功能及黄酮类化合物生物合成奠定基础,依据多穗柯转录组测序结果,设计特异性引物,PCR扩增得到4CL基因cDNA全长序列,利用相关软件进行生物信息学分析;采用qRT-PCR法检测4CL基因在不同器官中的表达情况,使用SPSS18.0软件分析其表达量与根皮苷含量间的关系。结果表明:多穗柯4CL基因cDNA全长1704bp,包含长1629bp的开放阅读框,编码含542个氨基酸的蛋白质。该蛋白定位于细胞质中,属亲水性蛋白。多穗柯4CL基因在除根以外的各个器官中均有表达,且表达量与根皮苷含量间呈极显著的正相关关系(P<0.01)。     

天女木兰种子天冬氨酸蛋白酶基因的克隆与分析

天冬氨酸蛋白酶在天女木兰种子萌发过程中发挥着重要作用。以天女木兰种子为试验材料,利用同源克隆法RACE技术,从天女木兰种子中克隆得到天冬氨酸蛋白酶基因,全长1 545 bp,可以编码514个氨基酸残基。同源性分析显示,目的序列推导的氨基酸序列与其它植物中已知的天冬氨酸蛋白酶基因的氨基酸序列的同源性都在85%以上;采用生物信息学的方法进行分析,初步预测该基因编码的蛋白属于跨膜的分泌蛋白,大部分区域属于疏水结构;结构域的预测分析显示,此类蛋白包含约100个植物组织蛋白酶所特有的区域,该区域可能在植物液泡中发挥着重要作用。本研究为下一步系统、全面地研究天女木兰种子休眠的分子机理奠定了基础。     

油茶乙酰辅酶A羧化酶BC亚基全长cDNA克隆及序列分析

以油茶‘湘林1号’品种的近成熟种子为材料,采用简并PCR、RACE、交错PCR等技术,获得了油茶乙酰辅酶A羧化酶BC亚基基因的全长cDNA克隆。该基因cDNA序列全长1 901 bp,含有一个1 599 bp的ORF,编码533个氨基酸残基。BC蛋白的等电点pI为6.88,分子量为58 509.3 u,含两个跨膜结构域,是一个不稳定的非分泌蛋白。模体搜索结果表明在BC上具有N-糖基化位点、蛋白激酶C磷酸化位点、ATP结合位点等多个功能结构域。同源建模的模型中发现12个α-螺旋,整个BC蛋白为一个内凹的结构。该基因已登录到GenBank,并被命名为co-bc。     

油桐乙酰辅酶A羧化酶BC亚基全长cDNA克隆及序列分析

以葡萄桐的近成熟种子为材料,根据油桐转录组测序结果设计引物,采用RT-PCR技术克隆了油桐乙酰辅酶A羧化酶BC亚基基因的全长c DN A序列。该基因c DNA序列全长1 605 bp,编码534个氨基酸。推导出的BC亚基氨基酸序列蛋白的等电点p I为7.98,相对分子量58 262.0 Da,稳定系数为38.13,生物信息学分析表明,此蛋白含有2个比较明显的跨膜区,是一个不稳定的非分泌蛋白。模体搜索结果表明在BC上具有ATP结合位点。三级结构中有16个α螺旋,3个部分形成一个内凹的结构。     

核盘菌arom基因的克隆与序列分析

本文用反向 PCR 方法扩增并分析了核盘菌 arom 基因,以研究核盘菌和其他真菌在该基因之间的进化关系,并为其编码蛋白(AROM)的催化机制研究、植物转基因和蛋白抑制剂设计提供基础。arom基因编码预分支酸芳香族氨基酸(苯丙氨酸、酪氨酸和色氨酸)合成途径中催化第二步到第六步的五功能多肽。序列分析表明核盘菌 arom 基因含有一个4773bp 的编码框,无内含子。推测的蛋白序列含有 1590 个氨基酸,与已知的所有 AROM 蛋白同源。理论分子量(Mw)和等电点(pI)分别是 172658.78D 和 6.45。核盘菌 arom 基因的GC%是 44.94%。根据搜索二级数据库 CDD 和 Prosite 的结果表明该 AROM 蛋白含有五个保守结构域:脱氢奎尼酸合成酶结构域、脱氢奎尼酸脱水酶(脱氢奎尼酸酶)结构域、莽草酸脱氢酶结构域、莽草酸激酶结构域、5 -烯醇丙酮酰莽草酸-3-磷酸合成酶(EPSP 合成酶)结构域和四个模式:两个 EPSP合成酶模式、Ⅰ型脱氢奎尼酸酶活性位点模式和莽草酸激酶模式。通过比对和参照 PIR 数据库显著位点规则发现核盘菌AROM 蛋白含有四个保守碱基。在基因 5′非翻译区-23 和-77位置分别存在 TATA 盒和 CAAT 盒。在基因下游发现有两个位点可能是 polyA 加尾信号。另外还分析了 arom 基因的系统发育。图 5 参 13。     

落叶松LaIAA2基因的克隆及表达分析

以落叶松扦插生根率存在显著差异的2个优良全同胞无性系茎和根为材料,利用抑制消减杂交(SSH)技术构建正、反2个落叶松扦插生根过程差别表达cDNA文库。序列分析获得1个AUX/IAA基因片段。运用Race技术,成功获得这个Aux/IAA基因的全长。同时,运用实时定量PCR和半定量PCR技术分析这个基因的表达谱,结果表明:LaIAA2基因在茎中高表达,叶中低表达。在生长素处理后,LaIAA2基因表达明显增加,表明这个基因受生长素调控。     

Purification of four strains of endophytic fungi from Astragalus and their optimized liquid fermentations

This study was designed to isolate endophytic fungi from A. mongholicus(growing in northeast China) to determine whether they can produce bioactive metabolites. Four strains of endophytic fungi(strains 16, 17, 23 and 75) were successfully isolated from A. mongholicus using the surface disinfection method. According to ITS-rDNA sequences analysis, strains 16 and 75 were identified as Fusarium oxysporum, and strains 17 and 23 were identified as Bionectria ochroleuca. We applied the Box-Behnken design(BBD) to optimize the liquid fermentation conditions and obtain the maximum cell dry weight(CDW) yield. Optimal parameters were obtained under the following experimental conditions: temperature of 28°C, potato dextrose agar(PDA) liquid medium of 80 mL and rotation speed of 150 rpm. The four isolated endophytic fungi did not produce astragalosides I–IV, flavonoids or polysaccharides. Isolation of additional species of endophytic fungi from A. mongholicus and determination of their capacity to produce biologically active substances are subjects in need of further research.