短波紫外线诱导采后番茄苯丙氨酸解氨酶的分离纯化

以番茄为试材,经2.4kJ/m2短波紫外线(UV-C)照射,取果皮经冰浴研磨、硫酸铵分级沉淀、凝胶过滤和离子交换层析,分离纯化苯丙氨酸解氨酶。经测定,紫外照射组PAL活性为81.0U/mL,比对照组高5U/mL,分离纯化倍数为16.4倍,纯化获得的酶经SDS—聚丙烯酰胺凝胶电泳分析,其亚基分子量约为76kD。     

番茄叶霉病抗性与苯丙氨酸解氨酶的相关性

以含对叶霉病不同抗性基因的番茄品种为材料，研究了叶霉病菌侵染过程中PAL酶活性和次生代谢物木质素含量的变化与品种抗性、病原菌生理小种间的相互关系. 种后苯丙氨酸解氨酶活性增加，抗病品种的PAL酶活性高峰出现早于感病品种。番茄品种的抗病性与其接种后36h PAL酶活性变化百分比呈极显著正相关（r=0.96^**)。另外，还成功地利用PAL酶活性变化速度这一指标对叶霉病生理小种的变异进行了生化鉴定。     

棉花苯丙氨酸解氨酶基因家族的生物信息学分析

【目的】苯丙氨酸解氨酶是苯丙烷类代谢的关键酶,对调节植物生长和抗逆有重要作用。从全基因组层次了解棉花苯丙氨酸解氨酶基因家族的进化和功能,可为相关研究提供数据支撑。【方法】利用HMMER和BLASTP从棉花基因组筛选苯丙氨酸解氨酶基因家族,利用MEGA-X进行进化分析,利用MCScanX进行共线性分析,并利用NCBI SRA的数据进行表达分析。【结果】本研究分别从陆地棉、海岛棉、亚洲棉和雷蒙德氏棉基因组中筛选到12、13、7、8个苯丙氨酸解氨酶基因,依据进化分析和共线性关系数据,我们推测四种棉花的二倍体祖先起初应有6个苯丙氨酸解氨酶基因。顺式作用元件分析结果表明,苯丙氨酸解氨酶基因家族含有较多的光响应、激素响应、胁迫响应和生长发育相关的元件。苯丙氨酸解氨酶的蛋白序列具有保守的基序。陆地棉的12个苯丙氨酸解氨酶在低温、高温、盐和PEG胁迫下有不同的表达方式。【结论】本研究明确了苯丙氨酸解氨酶在棉花基因组中的数量、理化性质、进化和表达特征,为苯丙氨酸解氨酶的深入研究提供了参考。     

桂花苯丙氨酸解氨酶基因的克隆与序列分析

利用一对简并引物,从桂花(Osmanthus fragrans)中克隆得到苯丙氨酸解氨酶(PAL)基因的cDNA片段,命名为OfPAL,GenBank登录号为GU991822.OfPAL长866 bp,编码288个氨基酸.通过核苷酸和蛋白质序列多重比较,发现OfPAL与其他植物的PAL基因高度同源.OfPAL编码的蛋白质序列包含与橄榄、烟草、辣椒PAL蛋白质相同的脱氨基位点和催化活性位点.PAL系统进化树显示,OfPAL与菊科植物的PAL基因亲缘关系较近.本研究通过克隆OfPAL基因,可为桂花对不良环境的抵御能力以及桂花类黄酮积累方面的研究奠定基础.     

苯丙氨酸解氨酶基因家族在大豆中的时空表达研究

苯丙氨酸代谢途径是植物最重要的次生代谢途径之一,其下游产物包括异黄酮、植保素、木质素等多种次生代谢产物。苯丙氨酸解氨酶（PAL）是苯丙氨酸代谢途径的起始酶和关键酶。PAL基因家族包含8个基因,为了明晰这8个基因在大豆植株内的表达情况,通过实时定量PCR研究了PAL家族所有成员在大豆植株内的时空表达差异性。发现PAL基因家族总体的相对表达量在生殖生长时期高于营养生长时期,在叶片和子粒中相对表达量较高。PAL1-1、PAL2-1和PAL2-3基因在各部位相对表达量明显高于其他同家族基因,PAL1-1基因各时期稳定表达,PAL2-1和PAL2-3基因在生殖生长后期先后出现表达峰值。这些结果表明大豆PAL基因家族各成员的相对表达量在时间和空间上存在差异,具有一定时空表达特异性。     

夹竹桃苯丙氨酸解氨酶的基因克隆与序列分析

首次从夹竹桃中克隆得到苯丙氨酸解氨酶(PAL)基因cDNA片段,并命名为NoPAL1,GenBank登录号为AY747677,这也是首次从夹竹桃花青苷代谢途径中克隆得到的基因。NoPAL1长866个bp,编码289个氨基酸。通过核苷酸和蛋白质序列多重比较发现NoPAL1与其他植物的PAL基因高度同源。NoPAL1编码的蛋白质序列包含与水稻、玉米PAL蛋白质相同的脱氨基位点和催化活性位点。PAL系统进化树表明,NoPAL1与乔木类植物的PAL基因聚类关系最近。克隆NoPAL1基因为利用基因工程技术来调控夹竹桃花青苷的代谢从而调控夹竹桃花的颜色提供了基因资源。     

夏枯草苯丙氨酸解氨酶基因的克隆与表达分析

苯丙氨酸解氨酶(PAL)是迷迭香酸合成途径中的关键酶之一,根据其他植物PAL基因的保守区域设计特异引物,利用3’-RACE-PCR技术,本研究首次从夏枯草中克隆得到了PAL基因的cDNA片段序列,命名为PvPAL,Gen-Bank登录号为JN65446。PvPAL基因cDNA片段长1 306 bp,其中编码区域为1 047 bp,编码349个氨基酸。蛋白质序列多重比较结果显示,PvPAL蛋白质序列与丹参、地黄、黄芩、藿香等植物的PAL蛋白质高度同源。PAL系统进化树分析结果表明,PvPAL与唇形科植物的PAL基因亲缘关系最近。组织表达分析结果显示,PvPAL基因在根、茎、叶中均表达,其中根中表达量最高。PvPAL基因的克隆为进一步研究夏枯草迷迭香酸合成的分子机制奠定了基础。     

玉米苯丙氨酸解氨酶家族基因的鉴定与纹枯病的抗病分析

玉米纹枯病是中国西南玉米产区的主要病害之一,苯丙氨酸解氨酶(PAL)活性变化在不同纹枯病抗性的玉米材料中存在差异,目前对PAL家族基因还缺乏系统深入的研究,尤其是参与玉米-纹枯病菌互作的研究甚少。本研究利用RT-qPCR方法,结合芯片数据库和转录组测序结果,鉴定了玉米PAL家族基因和组织表达特征,并分析了其在纹枯病菌诱导前后抗病和感病自交系中的表达情况。结果显示,玉米基因组中共有13个PAL基因,Zm PAL2、ZmPAL3、ZmPAL5、ZmPAL8、ZmPAL9和ZmPAL10均为组成性高表达,ZmPAL1、ZmPAL4、ZmPAL6和ZmPAL7均为组织特异性低表达,而同源度极高的ZmPAL11、ZmPAL12和ZmPAL13在所有检测组织中均不表达。在强致病力纹枯病菌诱导前,10个PAL基因的本底表达量在抗病和感病自交系的叶鞘中均无明显差异,但在诱导后都增强高表达,以应对纹枯病菌的侵染。这些结果说明不同抗性的玉米材料中可能存在类似但响应程度不同的PAL家族基因参与玉米-纹枯病菌互作的机制。     

甘蓝型油菜品系一些酶的活性与抗菌核病的关系

以6个抗病品种（系）M004、湘油15号、中R-888、中R-783、084、085和二个感病品种（系）98C04、甘油5号为材料,研究接种菌核病菌前后叶片中过氧化物酶（PO）、多酚氧化酶（PPO）、苯丙氨酸解氨酶（PAL）、超氧物歧化酶（SOD）的活性变化。结果表明：接种前,PO、PAL二种酶活性与油菜品种抗病性相关不显著,而PPO、SOD酶活性     

绿色棉苯丙氨酸解氨酶基因GhPAL1的克隆和实时定量表达分析

本研究以拟南芥(Arabidopsis thaliana)苯丙氨酸解氨酶(PAL)基因cDNA序列(AAC18870.1)为探针,在GenBank中与棉花EST进行tBLASTn比对,将同源性高的EST序列拼接后获得一条棉花PAL全长cDNA基因序列,以此序列为模板设计引物,通过RT-PCR扩增从绿色棉纤维分离出了一个绿色棉PAL基因全长cDNA序列,将该基因命名为GhPAL1(GenBank登录号:JN032297)。序列分析表明,该cDNA全长2347bp,含有一个编码721个氨基酸的开放阅读框。实时荧光定量PCR分析发现:该基因在绿色棉纤维中的表达模式与白色棉纤维相似,但两者在同一时期的表达量差异显著,其在花后6d绿色棉纤维细胞中的表达量是同期白色棉的6倍以上,从该基因的表达特征推测GhPAL1基因可能在绿色棉纤维色素合成过程中发挥重要作用。     

甘蓝型油菜亚基因组间杂种（ArAnCcCn）的细胞学和育性研究

以新型甘蓝型油菜（A^rA^rC^cC^c）与自然甘蓝型油菜品种湘油15（AⁿAⁿCⁿCⁿ）杂交，人工配制了A^rAⁿC^cCⁿ油菜亚基因组间杂种。亚基因组间杂种花粉母细胞减数分裂基本正常，中期I大多以19个二价体的构型存在，但偶尔也有单价体、三价体和四价体等异常情况出现。在减数分裂后期Ⅰ和后期Ⅱ，绝大多数细胞中的同源染色体和姊妹染色单体正常分离，仅在很小比例的花粉母细胞中观察到落后染色体。不同亚基因组间杂种的减数分裂正常程度存在一定的差别，其中Ar和An基因组之间的遗传分化可能是主要原因。产量分析表明亚基因组间杂种具有较强的杂种优势潜势，并且亚基因组间杂种的种子产量和正常花粉母细胞所占比例呈显著正相关。     

甘蓝型油菜与近缘种、属杂交时花粉-雌蕊相互作用的研究

甘蓝型油菜作母本与白菜、甘蓝、黑芥、芥菜型油菜和埃塞俄比亚芥杂交时,花粉粘合程度减弱。花粉管常常不能穿入柱头,与花粉管接触的柱头乳突细胞内普遍产生胼胝质。少数异源花粉管能穿过柱头进入花柱,但时有异常胼胝质沉积在花粉管中。海甘蓝的花粉很难粘合在甘蓝型油菜柱头上。异种花粉与甘蓝型油菜雌蕊的亲和性按大小依次     

Comparisons of Isozyme Patterns in Resynthesized Amphihaploid Rapeseed (Brassica napus) and Their Parental Species Brassica campestris and Brassica oleracea

Resynthesized rapeseed plants produced by embryo culture and the progeny of their selfed parents from Brassica oleracea and Brassica campestris were analyzed by starch gel electrophoresis staining of enzymes GPI, LAP and SDH. In at least one unequivocal case it could be proved for each enzyme system studied that the alleles of both parents arc independently expressed in the newly synthesized individuals and that in the pattern of the dimeric enzyme GPI interlocus bands of the homoeologous loci arc visible. Comparisons with enzyme patterns in cultivated B. napus leads to the conclusion that in general active enzyme loci of both parental genomes are present. This is of great significance in estimating slide frequencies, degree of heterozygosity and other genetic parameters of B. nupus populations/cultivars.     

Efficiency of a novel gametocide amidosulfuron on rapeseed (Brassica napus)

The efficiency of a novel gametocide amidosulfuron, 1-(4, 6-dimethoxypyrimidin- 2-yl) -3-mesyl (methyl) sulfamoylurea, was evaluated on rapeseed ( Brassica napus L.). Double application of 0.09–0.12 g/ha amidosulfuron at the uni-nucleate stage of longest buds and 12 days later, made 97.4–100% plants male-sterile in seventeen out of 20 cultivars tested. The shrunken anthers could not release pollen or produced only dysfunctional pollen grains that could not be stained by aceto carmine. The treatment also caused 10–30% reduction on seed setting in comparison to the controls. The purity of hybrid seed from crosses of 'C161 × Huaye' and 'Zhongshuang No.2 × Huaye' based on amidosulfuron was 99.8% and 100.0%, respectively. The results suggested that amidosulfuron is an efficient gametocide for B. napus .     

甘蓝型杂交油菜种子胎萌发生特性的初步研究

本试验以甘蓝型杂交种秦油２号及其三系种子为材料,于１９９２￣１９９３两年在浙江农业大学农场进行。试验结果表明,甘蓝型杂交油菜的杂种秦油２号,不育系陵２Ａ和保持系陕２Ｂ种子胎萌发生在开花后１９天左右,并随着种子发育日趋成熟而有不为断增多的趋势,而常规品种是Ｃ１种子胎萌发生在成熟后期（开花后３１天）。角果内位于果身中部的胎萌粒多于果身两端部。不同类型和品种,不同年份,具有胎萌特性同一植株的不同花序部位     

Identification and Genetic Studies of the Inhibition of Dominant Male Sterility in Brassica napus

By transferring dominant male sterility (DMS), caused by the gene Ms, to genotypes with various types of cytoplasm 12 DMS lines were developed and a number of crosses made between the DMS lines and other genotypes of Brassica napus. During the course of this population improvement programme, 16 genotypes were identified as having the capacity to restore the fertility of F₁ plants with the Ms gene. According to pedigree analysis, the inhibitory gene in those lines probably originated from a few genotypes from Australia and Germany. In further studies the inheritance of the sterility inhibition was determined, providing definite evidence that dominant male sterility and its inhibition in B. napus are controlled by two dominant interacting genes rather than by multiple alleles.     

Carbohydrate Dynamics in Leaves of Rapeseed (Brassica napus) Under Drought

Until now, the carbohydrate dynamics in leaves of rapeseed under drought have largely been unknown. For this reason, a growth chamber study was conducted to examine whether the accumulation of carbohydrates under drought stress contributes to osmotic adjustment in leaf tissue. Plants of the cultivar Titan were subjected to temporary drought in the vegetative and reproductive stages. A third variant of long‐term drought covered the period from leaf development to flowering. The level of sucrose decreased under moderate water deficit, but accumulated under severe long‐term drought. Concentrations of glucose, fructose and trehalose were significantly enhanced and that of raffinose decreased in all the variants of drought. There was no evidence that any of the carbohydrates analysed in this study or the activities of soluble acid and cell wall invertases contributed to a drought‐induced accumulation of osmolytes. The results of this study indicate that osmotic adjustment in response to drought in leaves of the rapeseed cultivar Titan is only limited. It is virtually impossible that carbohydrates function as osmoprotectants in leaves of this cultivar, rising above that of the frequently detected accumulation of proline in rapeseed under water deficit.     

Genetic analysis of four self-incompatible lines in Brassica napus

Reciprocal hybridization between four self-incompatible lines of Brassica napus: 271, 181, 184 and ‘White Flower’, revealed incompatibility. The reciprocal F₁s obtained by bud pollination showed self-incompatible reactions, and no segregation for self-incompatibility was observed in all the reciprocal F₂ populations, indicating that lines 271, 181, 184 and ‘White Flower’ were genetically identical with regard to self-incompatibility. Observations of self-incompatibility in 17 hybrids from crosses between line 271 and 17 varieties of B. napus showed 10 of the F₁ hybrids to be self-compatible, while four were partially self-compatible and three were self-incompatible. Genetic analysis based on F₂ and BC₁ populations from five self-compatible F₁ hybrids and two self-incompatible F₁ hybrids suggested the existence of at least two loci controlling the self-incompatibility of line 271: one is the S locus, with dominant and recessive relationships between the S alleles, and the other is the suppressor (sp) of the S locus. The sp locus is genetically different from the S locus, and also shows dominant and recessive relationships between the sp alleles.     

甘蓝型油菜遗传多样性及其与杂种表现的关系

以3个双低甘蓝型油菜自交不亲和系、 22个不同来源的父本品种及其按NCⅡ法配制的66个杂种为材料, 应用AFLP及RAPD标记技术研究亲本的遗传多样性及遗传距离与杂种产量、含油量的关系. 结果表明, 根据遗传距离可将3个自交不亲和系分为2组, SI-1300和SI-1310为一组, SI-1320单独为一组; 父本品种分为5组, 中国品种为1组, 国外品     

Mapping boron efficiency gene(s) in Brassica napus using RFLP and AFLP markers

Soil boron (B) deficiency, a world‐wide problem in agriculture, exists for more than 33 million hectares of cultivated land in China. Genetic variation exists for B‐efficiency among different Brassica napus germplasms. To identify genes controlling B efficiency, an F₂ population of B. napus was constructed from a cross between a high B‐efficiency cultivar, ‘Qingyou 10’, and a low B‐efficiency cultivar, ‘Bakow’, and then evaluated for seed yield, bolting date and maturity date under B‐deficient conditions. The ratio of high B‐efficiency to low B‐efficiency individuals fitted the expected ratio of 3:1, indicating a major gene controlling the B‐efficiency trait. The major gene was mapped in the ninth linkage group of B. napus. Genome‐wide quantitative trait loci (QTL) analyses detected one major locus near the major gene, which explained 64.0% of the phenotypic variance. At the same time, three minor loci in three linkage groups were also detected, and these minor loci individually explained 39.2%, 32.5% and 36.9%, respectively, of the phenotypic variance. A total of 11 QTLs were detected for bolting date and maturity date, some of which were associated with B‐efficiency.