家稗Pdk基因叶片特异性表达载体的构建及农杆菌的导入

【研究目的】构建含家稗Pdk基因的叶片特异性表达载体;【方法】通过PstI和SalI单酶切分别从质粒pRGN及pUCm-Pdk上获得Rubisco小亚基启动子和Pdk基因,将其连入表达载体pCAMBI1301, 并通过冻融法将重组质粒导入根癌农杆菌EHA105;【结果】构建了由Rubisco小亚基启动子调控的Pdk基因植物表达载体p1301-Prbs-Pdk,选择标记基因为Hpt（潮霉素磷酸转移酶基因）,经酶切和PCR鉴定,表达载体已成功导入农杆菌EHA105中;【结论】丙酮酸磷酸双激酶（PPDK）是C4光合途径中的关键光合酶,本实验中构建了含有rbcs启动子的适于单子叶植物转化的Pdk基因表达载体,为提高转基因植物光合效率奠定了基础。     

芍药等8种植物COMT基因的生物信息学分析

植物咖啡酸-O-甲基转移酶(caffeic acid O-methyltransferase,COMT)基因对木质素的合成具有重要的调控作用。本研究采用生物信息学方法对芍药等8种植物COMT基因编码的氨基酸序列进行比对,进而对其蛋白理化性质、信号肽、跨膜结构域、磷酸化位点、亚细胞定位、二级结构与三级结构进行了预测与分析。结果表明,芍药等8种植物COMT基因编码氨基酸的一致性可达65.91%,平均相对分子量为39437.79 Da,带有酸性等电点,含量最丰富的氨基酸为亮氨酸(Leu)和丙氨酸(Ala),无信号肽,无跨膜结构域,磷酸化位点数量最多的为丝氨酸和苏氨酸,定位于细胞骨架和细胞质中可能性最大,二级结构则以α-螺旋和无规则卷曲为主,三维结构具有一定的相似性。这一结果为今后深入开展植物COMT基因功能研究提供了科学依据。     

玉米高光效基因PPDK在籼稻IR64中的整合及其与光合作用相关的特性分析

丙酮酸磷酸双激酶（PPDK）在C4植物光合作用途径中,是CO2固定的关键酶。将编码玉米丙酮酸磷酸双激酶（PPDK）基因的质粒通过基因枪和农杆菌介导转化导入籼稻IR64中,PCR-Southern的结果表明,玉米丙酮酸磷酸双激酶（PPDK）基因已经整合到IR64中。在温室条件下,分析了转基因IR64植株的剑叶全氮含量,结果表明,大部分的转基因IR64植株剑叶的全氮含量高于非转基因IR64对照,在转基因IR64植株中,最高的剑叶全氮含氮量为3．61％,比非转基因的IR64对照高1．07个百分点,剑叶全氮含量提高了42．1％。对转基因IR64植株的产量构成因素的分析结果表明,不同的转基因IR64植株之间,产量构成因素差异比较大,比如植株干重、收获指数等,有助于育种家们选择不同的育种材料用于水稻育种。     

玉米PEPC基因和PPDK基因在籼稻明恢63中的整合及与光合作用相关的特性分析

丙酮酸磷酸双激酶（PPDK）是C4植物光合作用途径中,二氧化碳固定的关键酶。磷酸烯醇式丙酮酸羧化酶（PEPC）是在C4和景天酸（CAM）植物光合作用的初级碳同化中的一个关键酶,该酶介导不可逆的β羧化反应,将磷酸烯醇式丙酮酸转化成草酰乙酸和磷酸。本研究将编码玉米磷酸烯醇式丙酮酸羧化酶（PEPC）基因的质粒和编码玉米丙酮酸磷酸双激酶（PPDK）基因的质粒通过基因枪轰击转化的方法同时导入籼稻明恢63中,PCR-Southern印迹杂交的结果表明,玉米丙酮酸磷酸双激酶（PPDK）基因已经整合到明恢63中。在温室条件下,分析了转基因明恢63植株的剑叶全氮含量,结果表明,不同的转基因明恢63植株,其剑叶的全氮含量是不同的,大部分转基因明恢63植株剑叶的全氮含量高于非转基因明恢63对照的全氮含量,转基因明恢63植株ZHM3-50剑叶全氮含量为3．82％,比非转基因明恢63对照高0．45％。对转基因明恢63植株的产量构成因素的分析结果表明,在温室条件下,不同的转基因明恢63植株之间,产量构成因素差异比较大,比如植株干重、收获指数等。这些有助于育种家们选择不同的育种材料。     

家稗丙酮酸磷酸双激酶(PPDK)基因的克隆及序列分析

为揭示C4植物家稗[Echinochloa crusgalli var frumentacea(Roxb.)W.F.Wight] PPDK结构和功能特点,探索改善作物高光效途径,采用RT-PCR技术,克隆了家稗[Echinochloa crusgalli var frumentacea (Roxb.) W. F. Wight]丙酮酸磷酸双激酶（PPDK）基因的cDNA全长。核苷酸序列分析表明, 该片段全长3 085 bp, 包含一个2 844 bp的ORF,编码948个氨基酸(GenBank登录号：AY 792619)。编码区核苷酸序列与高粱（Sorghum bicolor）、甘蔗（Saccharum officinarum）、玉米（Zea mays）等C₄型pdk基因同源率分别为85.1%、84.0%和82.0%;与水稻[Oryza sativa (japonica cultivar-group)] pdk基因同源性也高达82.6%。氨基酸序列进化树分析表明其与玉米、水稻等禾本科植物最为接近;半定量RT-PCR研究表明,pdk基因仅在绿色叶片中表达。     

广东紫珠matK基因的生物信息学分析

通过PCR扩增获得广东紫珠matK基因的完整核苷酸序列,利用生物信息学对matK基因进行开放阅读框、氨基酸组成、蛋白质跨膜结构区、亚细胞定位和结合区分析,预测信号肽、氨基酸亲/疏水性、氨基酸及高级结构预测,采用UPGMA算法对13种紫珠属植物构建系统进化树。结果表明,广东紫珠matK基因序列长度为1 524 bp,编码507个氨基酸,有10个开放阅读框;matK基因编码的成熟酶K蛋白含20种氨基酸,二级结构中α螺旋占49.11%、β转角4.45%、无规则卷曲28.21%,为不稳定亲水性蛋白;无信号肽、无跨膜结构,定位于叶绿体亚细胞器。系统进化聚类结果表明,广东紫珠与紫珠、Callicarpa mollis的亲缘关系较近。     

稗草EcHPPD基因的克隆及生物信息学分析

旨在克隆稗草对羟基苯基丙酮酸双氧化酶(HPPD)基因为探究其基因功能提供基础,从而更好地开发除稗剂。以稗草叶片为材料,用同源克隆和c DNA末端快速克隆(RACE)技术克隆Ec HPPD基因序列,用生物信息学软件分析其特征。Ec HPPD基因的编码序列全长1317 bp,其编码蛋白质由439个氨基酸组成,相对分子质量为46.659 k D,等电点为5.30。稗草与同处禾本科的Dichanthelium oligosanthes(OEL23409.1:8-249)的HPPD蛋白进化程度最为相近。Ec HPPD三级建模蛋白结构由两条链构成,有2个Fe²⁺结合位点,并能与吡唑特活性中心骨架通过氢键相互作用。Ec HPPD可能与HGO、HPT1、TAT3、AT5G36160、TAT7等11个蛋白产生互作。本研究报道了Ec HPPD基因序列并对其进行了生物信息学分析,为创制新的HPPD抑制剂提供理论依据。     

无核白葡萄多酚氧化酶基因的克隆与序列分析

为获得无核白葡萄中多酚氧化酶(PPO)的基因序列,以无核白葡萄果实为试材,进行基因同源克隆,得到多酚氧化酶基因CDS全长序列,其完整的编码框为1 824 bp,编码607个氨基酸(GenBank登陆号为KP271928);系统进化分析可知,其与葡萄科植物PPO基因同源性最高;生物信息学方法发现该蛋白分子量为67 392.44 Da,等电点为6.42,具典型的酪氨酸家族结构域,无信号肽结构,但有一个跨膜结构域,为亲水性蛋白;二级结构预测发现,PPO具有CuA与CuB两个结合区,分别嵌入蛋白活性腔中。该研究为进一步从分子水平探讨多酚氧化酶与褐变的关系奠定理论基础。     

乙脑病毒Henan-09-03株E蛋白结构与功能生物信息学分析

利用生物信息学软件对Henan-09-03株乙型脑炎病毒(JEV)病毒E蛋白(囊膜蛋白)进行生物信息学分析、预测,为研究JEV的E蛋白基因工程疫苗提供依据。结果表明:E蛋白的分子质量为53 k Da,等电点是7.75,消光系数是66 515,不稳定系数为27.05,疏水平均值为-0.007,无信号肽,有2个跨膜区、27个蛋白修饰位点、5个二硫键、15个蛋白质结合位点,二级结构为α-β混合型、三级结构以同源四聚体形式存在。     

白羊草BiCDPK基因保守区的克隆及序列分析

为了研究CDPK基因的结构和功能,利用RT-PCR方法从白羊草中克隆得到BiCDPK基因,并对其进行生物信息学分析。结果表明：该BiCDPK基因cDNA长1048 bp,包含387 bp的开放阅读框,编码128个氨基酸。蛋白表现为弱酸性,且稳定、亲水,二级结构中以无规则卷曲和α-螺旋为主。亚细胞定位于细胞质中,无跨膜结构域,无信号肽。蛋白序列中存在1个丝氨酸磷酸化位点和3个苏氨酸磷酸化位点。进化分析结果表明,克隆的BiCDPK基因与玉米、小麦和水稻具有较高同源性,相似度分别是97%、86%、86%。为进一步研究BiCDPK基因的功能奠定了基础。     

南美蟛蜞菊毛状根乙醇提取液对种子萌发的影响

摘 要：【研究目的】本文拟通过用不同浓度的蟛蜞菊毛状根的乙醇提取液浸泡三叶鬼针草(Bidens pilosa L.)等种子研究其化感作用,为今后开展通过南美蟛蜞菊离体培养毛状根来生产高效低毒的“生物农药”及其应用奠定实验和技术基础。【方法】我们用0.5 g/mL、1.0 g/mL两种浓度蟛蜞菊毛状根的乙醇提取液,分别浸泡三叶鬼针草(Bidens pilosa L.)等种子3h、12h和24 h后,进行种子萌发实验。以清水浸泡后的种子萌发情况作为对照相比。【结果】提取液的浓度越高或浸泡时间越长,种子的萌发率越低,而且发现在0.5 g/mL(鲜重)的低浓度提取液中浸泡12 h以上,鬼针草、马唐和狗尾草已经不能正常萌发,在1.0 g/mL(鲜重)的高浓度提取液中浸泡12 h以上对种子的抑制作用更明显,鬼针草、马唐、稗草、狗尾草、蒲公英种子均不能萌发。而且发现无论是用0.5 g/mL(鲜重)的低浓度提取液还是用1.0 g/mL(鲜重)的高浓度提取液进行浸泡,浸泡时间在24 h以上,抑制效果就更加明显。【结论】结论：蟛蜞菊毛状根具有较强的化感作用。     

菊花桃不同器官除草活性的研究

明确菊花桃不同器官对生菜、黄瓜、反枝苋、苘麻、小麦和稗6种受体植物的除草活性,为进一步开展活性化合物的研究提供依据;在10g/L的添加浓度下,菊花桃的不同器官粉末对各受体植物幼苗的生长均具有不同程度的抑制作用,且普遍对胚根(或种子根)生长的抑制效果高于对胚轴(或胚芽鞘)生长的抑制。其中,以根、茎和花器官的总体抑制活性最强,而叶器官的效果则相对较低,6种受体植物中以反枝苋的敏感性最高,而稗的敏感性相对较低。说明菊花桃的根、茎和花等器官中均含有高除草活性物质,且对受体植物具有一定的选择性。     

Major Gene Plus Polygene Genetic Analysis of Lint Percent in Upland Cotton

[Objective] Lint percentage is a critical factor in cotton (Gossypium hirsutum L.) yield; however, there is limited genetic researches available to dissect the genetic characteristics of cotton lint percentage or for illustrating the mechanism of how lint percentage contributes to yield formation.【Methods】This study used the major gene plus polygene genetic model to perform a comprehensive study on genetic variation among lint percentage characteristics. In total, a 250-recombinant inbred line population was constructed for cultivar CCRI 70, a national high-quality cotton, and this population and the cultivar’s parental lines were phenotyped in nine environments in the Yellow River Valley, the Yangtze River Valley and Northwest Inland Region. 【Results】The lint percentage of the female parent sGK Zhong 156 was greater than that of the male parent 901-001 when grown in the nine environments. The lint percentage range of the recombinant inbred lines was 33.91%–40.18%, with an average of 38.01%, and the average was lower than that of the parental values. Thus, the performance was heterotic. The absolute values of skewness and kurtosis were less than 1.0, indicating a normal distribution, and the coefficients of variation ranged from 5.36% to 8.17%. Lint percentage showed the following geographical trend: Northwest Inland Region＞the Yellow River Valley＞the Yangtze River Valley. The genetic model is as follows: in different environments, lint percentage is controlled by two to four pairs of main genes or two pairs of major genes plus polygenes. The heritability of major genes ranged from 1.26% to 83.13%, the multiple gene heritability ranged from 27.35% to 90.83%, and the heritability of major genes plus multiple gene ranged from 92.00% to 99.35%. In the current study, lint percentage was mainly controlled by the major genes model having two major genes plus polygenes and the major gene model having two to four major genes. Among 2015 in Anyang, Henan, 2016 in Anyang, Henan and 2016 in Linqing, Shandong, only the former had four pairs of major genes. 【Conclusion】When the main gene plus polygenic benefits on lint traits is greater than 90%, then a high quality cotton is produced. The results are helpful in elucidating the genetic rules regulating the yield components of CCRI 70, and they provide a theoretical basis for improving cotton yield.     

二倍体野生棉与四倍体栽培棉基于GISH的遗传关系分析

[目的]研究二倍体野生棉与四倍体栽培棉间的遗传亲缘关系,进一步探索各棉种间的起源与进化.[方法]以5个二倍体基因组的代表种B1(异常棉)、C1(斯特提棉)、E2(索马里棉)、F1(长萼棉)以及G1(比克氏棉)的基因组DNA(gDNA)为探针,以2个四倍体栽培种(陆地棉中棉所16、海岛棉新海7号)有丝分裂中期染色体为靶DNA,进行了基因组原位杂交(Genomic in situ hybridization,GISH)分析.[结果]以B1、E2和F1gDNA为探针时,杂交信号主要分布在2个栽培种较长的13对A亚组染色体上;各产生3对较强的GISH-NOR信号,其中1对分布在较长的A亚组上,2对分布在较短的D亚组上,其GISH-NOR信号强度与分布情况与以D基因组棉种为探针时相似.说明二倍体B、E、F基因组与四倍体棉A亚基因组具有较高的同源性,亲缘关系更近.这一点与它们的地理分布情况相符;而它们基因组中的45S rDNA重复序列与二倍体D基因组的45SrDNA重复序列同源性较高.C1和G1中以gDNA为探针时,杂交信号分布在2个栽培种全部26对染色体上,无法区分开A或D亚组染色体,都有3对较强的GISH-NOR信号.这一现象与D基因组拟似棉(D6) gDNA为探针的GISH相似,表明二倍体C和G基因组与四倍体棉的A和D亚基因组均具有较高的同源性,或者C和G基因组同时含有A基因组(或其他非洲棉基因组)和D基因组成分,进一步证实了其基因组成分的杂合性;而它们基因组中的45S rDNA重复序列同属D基因组类型.[结论]这些发现可为棉花杂交育种和棉属起源与演化研究提供有用信息.     

蒲公英二倍体与四倍体的几个生理指标比较

【研究目的】通过测定和比较二倍体和四倍体蒲公英不同部位(根、叶、花序)的几个生理指标,包括类黄酮、异黄酮、Vc、可溶性糖含量、SOD、POD和CAT,探索药用蒲公英倍性育种的应用前景,为药用植物的多倍体诱导提供理论支持。【方法】以人工选育的四倍体药蒲公英为材料,以二倍体多裂蒲公英为对照,采用相关的方法进行分别测定。【结果】四倍体药蒲公英的这些指标比二倍体蒲公英都有所增加,其中类黄酮平均增加46%,异黄酮平均增加38%,Vc平均增加54%,可溶性糖平均增加50%,SOD平均增加29%,POD平均增加45%,CAT平均增加40%。【结论】人工四倍体药蒲公英的这些生理指标显示出较强的抗性基础,可望直接利用或作为进一步培育药蒲公英新品种的良好育种材料。     

Genome-Wide Identification and Expression Analysis of TLP Genes in Gossypium hirsutum L.

[Objective] Thaumatin-like proteins (TLPs) are important pathogenesis-related proteins that function during disease defense-related responses, participating in the defense reactions triggered by several kinds of pathogen infections. A genome-wide analysis of TLP genes could help increase our understanding of their molecular mechanism in response to Verticillium dahliae infections in cotton (Gossypium hirsutum L.). [Method] The TLP family members in the genomes of the G. hirsutum L. were identified and expression analyses of TLP genes in response to Verticillium dahliae inoculation were conducted using bioinformatics and real-time fluorescent quantitative PCR methods, respectively. [Result] In total, 88 TLP genes were identified in G. hirsutum. These TLPs were classified into 10 groups based on their amino acid sequences and a phylogenetic analysis. A gene structure analysis revealed that the number of exons ranges from 1 to 5, and the number of introns ranges from 0 to 4. TLP genes in the same group shared similar structures. Additionally, most TLP proteins contain five motifs that are arranged in the following order: 5, 4, 2, 3 and 1. In total, 87 TLP genes are distributed on 20 of the 26 chromosomes, and 42 and 45 TLP genes are distributed in the A and D subgroups, respectively. The real-time fluorescent quantitative PCR analysis showed that the expression levels of six candidate TLP genes were induced in both tolerant cotton cultivar GZ-1 and susceptible cotton cultivar 86-1, and showed a higher expression in the former. [Conclusion] These results provide a foundation for future studies of the functions and regulatory mechanisms of TLP family genes.     

枸杞LbCO基因的克隆与生物信息学分析

CONSTANS(CO)基因是高等植物叶片中光周期途径的关键成分,生物钟及光信号控制CO基因的表达。本研究以宁夏枸杞(Lycium barbarum L.)品种之一‘宁杞7号’叶片为实验材料,通过转录组测序结果筛选,利用RT-PCR技术扩增得到‘宁杞7号’的CO基因cDNA全长序列,命名为LbCO。利用生物信息学手段对所获得的序列进行结果及功能预测,结果显示:LbCO基因的cDNA序列全长1224 bp,编码407个氨基酸,分子质量为44.83 k D,理论等电点pI=5.58,不稳定指数为39.66,是一种稳定的非分泌蛋白。该蛋白亚细胞定位于细胞核,二级结构中无规则卷曲占比最高(54.48%),其次为α-螺旋(28.99%)。系统进化分析表明枸杞LbCO蛋白与其它物种的CO蛋白具有较高的同源性,其中与辣椒属CO蛋白亲缘关系最近。本研究为后续进一步探讨该基因编码的蛋白在调控枸杞花期研究方面提供理论参考。     

Susceptibility to sharka (Plum pox virus) in Prunus mandshurica×P. armeniaca seedlings

Prunus mandshurica [(Maxim.) Koehne] from Central Asia is a species related to apricot and grown in China and Mongolia. This species has been used in apricot breeding as a source of frost resistance. In addition, P. mandshurica has been suggested as the possible origin of some North American apricot cultivars resistant to sharka (Plum pox virus, PPV). The aim of this work was to transmit the resistance to PPV from P. mandshurica to the Spanish apricot cultivar ‘Currot’ by traditional crossing. The resistance to a Dideron PPV isolate of the descendants of P. mandshuricaבCurrot’ and their progenitors was evaluated under controlled conditions in a greenhouse. The results showed the susceptibility of both, the progenitors and the offspring to PPV, as being much higher than in other apricot seedlings. The results showed that the P. mandshurica accession studied is not a good progenitor in breeding apricot for PPV resistance, but just the opposite. The possible role of P. mandshurica as a source of resistance in apricot resistant cultivars is questioned.     

Genetic diversity among tea cultivars from China, Japan and Kenya revealed by ISSR markers and its implication for parental selection in tea breeding programmes

Tea plant [Camellia sinensis (L.) O. Kuntze] is an important beverage crop in the world. In recent years many clonal tea cultivars have been released, and they play major roles in improving the production and quality of tea. It is important to understand the genetic diversity and relatedness of these cultivars to avoid inbreeding and narrow genetic basis in future tea breeding. In the present study, genetic diversity and relationship of 48 tea cultivars from China, Japan and Kenya were evaluated by inter‐simple sequence repeat (ISSR) markers. A total of 382 ISSR bands were scored, of which 381 (99.7%) were polymorphic. The ISSR primers showed high ability to distinguish between tea cultivars according to their high Resolving Power (R_P) with an average of 7.4. The mean of Nei’s gene diversity (H) and Shannon’s information index (I) were 0.22 and 0.35, respectively. More abundant diversity was revealed among cultivars in China than those in Japan and Kenya. Within Chinese populations, the level of diversity in east China was higher than that in other regions. The coefficient of genetic differentiation (G_ST) was 0.202, which indicates a high degree of genetic variation within populations. This result was further confirmed by analysis of molecular variance, which revealed the variance component within the populations (92.07%) was obviously larger than that among populations (7.93%). The level of gene flow (N_m) was estimated to be 2.0. This could be explained by frequent natural cross‐pollination and seed dispersal among tea populations. The pairwise similarity coefficient between the cultivars varied from 0.162 to 0.538. A dendrogram of 48 tea cultivars was constructed where all the tested cultivars were divided into two groups. Our data show that the genetic relationship among tea cultivars can be determined by the ISSR markers. This will provide valuable information to assist parental selection in current and future tea breeding programmes.