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Characterization of a myrosinase cDNA from Brassica parachinensis and its defense role against Plutella xylostella after suppression

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Insect Science 2012年第4期19卷 461-471页

作者： Guang Yang Ding-Feng wang Zhi-Quan Dong Qian-Liang wang gefu wang-pruski Min-Sheng You lnstitute of Applied Ecology Fujian Agriculture and Forestry UniversityFuzhou Tea Research Institute Fujian Academy of AgriculturalScienceFu'anFujian Institute of Prevention and Control of Professional Diseases FuzhouChina Department of Plant and AnimalSciences Nova Scotia Agricultural CollegeTruroNova ScotiaCanada

In Brassicaceae, myrosinase catalyzes the hydrolysis of glucosinolate and plays an important role in anti-herbivore defense. We have cloned and characterized the full- length complementary DNA of myrosinase gene from Brassicaparachinensis that exhibits high sequence identity with myrosinase genes from other Brassica species. To investigate the role of this myrosinase in defense against the diamondback moth （Plutella xylostella）, we constructed an RNA-interference （RNAi） cassette expressing a double-stranded RNA that targeted myrosinase and transfected it into B. parachinensis. Myrosinase was sup- pressed in the resulting transgenic plants. Diamondback moth larvae feeding on transgenic plants had lower larval and pupal weights, longer pupal duration, and lower fecundity than those feeding on non-transgenic plants, suggesting that the diamondback moth has adapted to the glucosinolate-myrosinase defensive system. Therefore, the suppression of myrosinase is a potential approach for controlling the diamondback moth.

关键词： Brassica parachinensis myrosinase Plutella xylostella RNA interference

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芥蓝SPL基因家族鉴定及表达分析

西北植物学报 2022年第3期42卷 361-370页

作者：曾东琳唐为玲曾家晶 gefu wang-pruski 赖钟雄郭容芳福建农林大学园艺植物生物工程研究所福州350002 福建农林大学戴尔豪西大学联合实验室福州350002

SPL存在于所有绿色植物中,在植物生长发育及叶片形成中起着重要的作用。该研究以‘改良香菇’芥蓝为材料,采用生物信息学方法,对芥蓝SPL家族(BoSPL)进行全基因组鉴定及分析;采用PCR技术克隆BoSPL3-1、BoSPL10-2和BoSPL11-2基因,并对芥... 详细信息

SPL存在于所有绿色植物中,在植物生长发育及叶片形成中起着重要的作用。该研究以‘改良香菇’芥蓝为材料,采用生物信息学方法,对芥蓝SPL家族(BoSPL)进行全基因组鉴定及分析;采用PCR技术克隆BoSPL3-1、BoSPL10-2和BoSPL11-2基因,并对芥蓝不同生长时期及不同组织部位进行定量分析,为揭示‘改良香菇’芥蓝叶片发育过程的分子机制以及培育芥蓝新品种奠定基础。结果表明:(1)BoSPL家族共有31个成员,编码区蛋白质氨基酸长度在157~1028 aa之间,BoSPL蛋白质的氨基酸均没有信号肽,属于非分泌蛋白;亚细胞定位预测结果显示,所有的BoSPL家族成员均有定位在细胞核上,少数成员有定位在液泡、叶绿体和细胞质中;BoSPL家族基因包含内含子数量为1~9个,所有成员均包含SBP保守结构域;系统进化分析显示,BoSPL可以分为8个进化群亚组,各群基因数量不等,且BoSPL进化关系与AtSPL相似。(2)成功克隆到BoSPL3-1、BoSPL10-2和BoSPL11-2基因,并成功构建pCAMBIA1302-35S-BoSPL3-1-GFP、pCAMBIA1302-35S-BoSPL10-2-GFP和pCAMBIA1302-35S-BoSPL11-2-GFP重组质粒,经根癌农杆菌GV3101介导瞬时转化烟草下表皮细胞,激光共聚焦显微镜检测表明,BoSPL3-1、BoSPL10-2和BoSPL11-2均定位在细胞核中,与预测结果一致。(3)qRT-PCR分析显示,BoSPL3-1、BoSPL10-2和BoSPL11-2在‘改良香菇’芥蓝叶片中相对表达量均较高,但3个基因在叶肉中的相对表达量无明显差异,而BoSPL10-2在花状变形叶中却显著高表达,推测其可能与花状变形叶形成相关。

关键词：芥蓝 BoSPL 全基因组鉴定亚细胞定位

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芥蓝TCP家族全基因组鉴定及表达分析

中国农业大学学报 2022年第8期27卷 76-92页

作者：曾家晶赵怡娇曾东琳唐为玲李尊文 gefu wang-pruski 赖钟雄郭容芳福建农林大学园艺植物生物工程研究所福州350002 福建农林大学戴尔豪西大学联合试验室福州350002

为明确芥蓝TCP家族相关基因在叶片发育中的功能,基于甘蓝基因组数据,分析鉴定出芥蓝TCP基因家族有40个成员,参考拟南芥同源TCP基因注释及拟南芥数据库基因的相对表达量,初步选取BoTCP21和BoTCP25基因,采用qRT-PCR分析。结果表明:BoTCP21... 详细信息

为明确芥蓝TCP家族相关基因在叶片发育中的功能,基于甘蓝基因组数据,分析鉴定出芥蓝TCP基因家族有40个成员,参考拟南芥同源TCP基因注释及拟南芥数据库基因的相对表达量,初步选取BoTCP21和BoTCP25基因,采用qRT-PCR分析。结果表明:BoTCP21和BoTCP25均在叶片中大量表达,但二者在真叶不同部位的表达模式存在差异。为了进一明确TCP基因家族中参与叶片发育的基因,采用生物信息学方法并结合qRT-PCR对BoTCP家族进行了全面分析。结果显示:40个BoTCP都属于非分泌亲水性蛋白,分别定位在8条染色体上;系统进化树构建和亲缘关系分析将芥蓝中的BoTCP成员归为3类,其中PCF亚家族有19个成员,CYC亚家族8个成员,CIN亚家族13个成员。qRT-PCR结果表明:BoTCP21和BoTCP25在真叶和薹叶中具有较高的表达量,BoTCP14在开花结荚的植株根部表达量较高,而BoTCP16则在抽薹植株的根部表达量最高,说明BoTCP家族成员在组织部位表达存在时空特异性且广泛参与了植株的形态建成和器官发育。

关键词：芥蓝 TCP基因家族全基因组鉴定生物信息学表达分析

Rapid regeneration of stable transformants in cultures of potato by improving factors influencing Agrobacterium-mediated transformation

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Advances in Bioscience and Biotechnology 2010年第5期1卷 409-416页

作者： Bipasha Chakravarty gefu wang-pruski 不详

An efficient and rapid Agrobacterium tumefaciens-mediated transformation protocol was developed to generate activation-tagged mutant lines with the aim of large-scale functional analysis of the potato genome. The explants were inoculated with an Agrobacterium strain harboring the binary plasmid pSKI074 containing four CaMV 35S enhancers in the T-DNA region which activates the downstream genes in the host plant after its integration. Various parameters investigated to increase transformation efficiency were the type and age of explant, cultivar, hormone combinations, preculture of explants, period of co-cultivation with bacteria and concentration of bacterial cultures used for transformation. Stem explants from 5 week old plantlets of cv. Bintje which had undergone phytohormone pretreatment for 4 days, inoculation with diluted bacterial concentration of OD600 = 0.2 containing acetosyringone followed by 2 days of co-cultivation and selection in media with IAA and trans-zeatin all helped in greatly improving the transformation efficiency. The total time required from infection to rooted shoots was 6-7 weeks. Initial evidence for stable integration and expression of the transgenes by PCR analysis showed that over 93% of the regenerated lines were transgenic and this was confirmed by Southern hybridization.

关键词： Bacterial Concentration Cultivar Explant Potato Transformation Preculture

两轮多重反转录巢式PCR方法扩增全长GalUR基因(英文)

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农业生物技术学报 2005年第6期13卷 728-732页

作者：杨静慧 gefu wang-pruski Mark Hodges 天津农学院园艺系 Nova Scotia农学院植物动物科学系加拿大农业食品研究院园艺研究中心

研究了两轮多重反转录巢式PCR扩增全长基因的方法。先用多重巢式引物和cDNA进行第1轮PCR扩增;再用纯化的第1轮PCR产物和相同的引物进行第2轮扩增;最后通过改变Mg2+浓度、退火时间和退火温度进行PCR条件的优化,以增加PCR扩增的产量,用于... 详细信息

研究了两轮多重反转录巢式PCR扩增全长基因的方法。先用多重巢式引物和cDNA进行第1轮PCR扩增;再用纯化的第1轮PCR产物和相同的引物进行第2轮扩增;最后通过改变Mg2+浓度、退火时间和退火温度进行PCR条件的优化,以增加PCR扩增的产量,用于克隆。结果显示该方法方便和快速地扩增出了草莓(***)高产量和高质量的全长GalUR基因,也非常快速地扩增出了用于基因构建的带有酶切位点的全长GalUR基因。该方法也可以用于其它全长基因的克隆。

关键词：两轮多重扩增巢式PCR 全长基因 GalUR

Metabolomics Profile of Potato Tubers after Phosphite Treatment

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American Journal of Plant Sciences 2018年第4期9卷 845-864页

作者： Xingxi Gao Steven Locke Junzeng Zhang Jyoti Joshi gefu wang-pruski Department of Plant Food and Environmental Sciences Faculty of Agriculture Dalhousie University Truro Canada Department of Edible Fungi Science Faculty of Agriculture Ludong University Yantai China Aquatic and Crop Resource Development Research Centre National Research Council of Canada Charlottetown Canada Aquatic and Crop Resource Development Research Centre National Research Council of Canada Halifax Canada

Phosphite (Phi)-based fungicides are used to control the oomycete Phytophthora infestans which causes late blight disease, the most devastating disease in potatoes. In order to examine the effects of Phi-based fungicides on potato tubers through foliar or post-harvest application, a metabolite profiling approach based on gas chromatography coupled to mass spectrometry (GC-MS) has been established. A total of 132 metabolites were detected using the GC-MS approach. Among these, 34 metabolites were identified after normalization and annotated with a compound name with standard mass spectral library. Metabolomic analysis of Phi-treated plants showed significant differences in the levels of many metabolites especially amino acids. Multivariate statistical approaches, such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were employed to explore the relationships between metabolites to detect group differences. A good discrimination between the control and the Phi-treated plants was observed, which demonstrated that significant changes in the metabolite profile have been caused by the two different Phi applications (foliar or post-harvest). This finding suggests that the alteration of specific metabolite levels by accumulation of Phi can lead to resistance against the pathogen.

关键词： Potato Metabolomics GC-MS Phosphite Phytophthora infestans

马铃薯HSP17.7基因的克隆及其对高温逆境的响应

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西北植物学报 2019年第11期39卷 1952-1960页

作者：奚玉培张志忠王景荣韩晓云 gefu wang-pruski 福建农林大学园艺学院福建农林大学-戴尔豪西大学联合实验室福州350002 戴尔豪西大学农学院加拿大特鲁罗B2N 5E3

热激蛋白和植物对高温的响应密切相关,同时在植物生长发育调控和逆境抵抗等方面具有重要作用。该研究克隆了马铃薯热激蛋白基因StHSP 17.7(GenBank登录号为XP_006350804.1),对其全长cDNA序列进行了相关生物信息学分析,并利用qRT-PCR分析... 详细信息

热激蛋白和植物对高温的响应密切相关,同时在植物生长发育调控和逆境抵抗等方面具有重要作用。该研究克隆了马铃薯热激蛋白基因StHSP 17.7(GenBank登录号为XP_006350804.1),对其全长cDNA序列进行了相关生物信息学分析,并利用qRT-PCR分析StHSP17.7基因在高温胁迫下的差异表达特性。结果显示:(1)StHSP 17.7全长755 bp,包含465 bp开放阅读框,编码154个氨基酸。(2)StHSP17.7分子质量约为17.62 kD,等电点7.91,为亲水性蛋白,C端含有保守序列Ⅰ和Ⅱ组成的ACD结构域,属于典型的sHSPs家族成员。(3)系统进化分析发现马铃薯小分子热激蛋白归为12个亚家族,其中StHSP17.7与马铃薯HSP17.6蛋白亲缘关系较近,属于小分子热激蛋白C的Ⅰ类家族成员;基因结构分析显示,在马铃薯48个sHSP基因中,StHSP17.7基因不含内含子,含1个内含子的基因有23个,占47.9%。(4)qRT-PCR分析显示,高温能够快速诱导StHSP 17.7基因表达,且基因表达量呈爆发式变化,在24 h达到最高值。研究表明,StHSP 17.7基因明显参与了马铃薯对高温的响应。

关键词：马铃薯 StHSP17.7基因高温逆境小分子热激蛋白

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芥蓝miR156a家族进化特性及表达分析

西北植物学报 2020年第8期40卷 1313-1322页

作者：陈冰星陈泽源赵怡娇唐为玲 gefu wang-pruski 郭容芳福建农林大学园艺学院福建农林大学-戴尔豪西大学联合实验室福州350002 福建农林大学园艺学院园艺植物生物工程研究所福州350002 戴尔豪西大学农学院植物食品和环境科学系加拿大特鲁罗NSB2N5E3

miRNA广泛参与植物的发育过程。芥蓝形态多样,叶片发育因品种而异。为了解miR156a家族的进化特性及其在芥蓝叶片发育中的表达模式。该研究对芥蓝miR156a成员及其前体pre-miR156a成员进行生物信息学分析,比较不同品种芥蓝叶形的差异,并采... 详细信息

miRNA广泛参与植物的发育过程。芥蓝形态多样,叶片发育因品种而异。为了解miR156a家族的进化特性及其在芥蓝叶片发育中的表达模式。该研究对芥蓝miR156a成员及其前体pre-miR156a成员进行生物信息学分析,比较不同品种芥蓝叶形的差异,并采用qRT-PCR方法分析芥蓝不同组织部位中pre-miR156a的表达水平、以及叶形相似的芥蓝品种‘翠宝’和‘改良香菇’中不同类型叶片的pre-miR156a成员及其靶基因的表达水平。结果表明:(1)多序列比对和进化树分析发现芥蓝miR156a家族成员和pre-miR156a-3p_1在进化过程中高度保守;二级结构预测发现pre-miR156a每个成员均能形成茎环结构并包含2~3个miR156a成员序列;靶基因预测显示miR156a-5p和miR156a主要靶向SPL,而miR156a-3p_1则靶向CTPS等不同的基因。(2)‘翠宝’和‘改良香菇’芥蓝的叶形最为相似,qRT-PCR分析显示,pre-miR156a在‘翠宝’营养生长期的叶片中高度表达。(3)pre-miR156a成员在‘翠宝’和‘改良香菇’不同类型叶片中差异表达,pre-miR156a主要在成熟叶和菇叶中表达,而pre-miR156a-3p_1和pre-miR156a-5p在第一片真叶中表达量更高。(4)靶基因分析的结果在不同品种中呈现不同趋势,‘翠宝’成熟叶中SPL10/15高表达,SPL2表达量降低;‘改良香菇’中SPL10在成熟叶和菇叶中表达降低,SPL15在菇叶中高表达,SPL2在不同类型叶片中表达无差异。研究认为,miR156a成员及其靶基因SPL2/10/15可能参与调控芥蓝叶片发育,不同品种中作用的靶基因可能存在差异,从而导致了各品种芥蓝叶片发育的差异。

关键词：芥蓝叶片菇叶 miR156a SPL

DNA Markers for Selection of Late Blight Resistant Potato Breeding Lines

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American Journal of Plant Sciences 2017年第6期8卷 1197-1209页

作者： Shaohua Chen Tudor Borza Bohyun Byun Robert Coffin Joyce Coffin Rick Peters gefu wang-pruski Department of Plant Food and Environmental Sciences Faculty of Agriculture Dalhousie University Truro Canada Privar Farm Inc. North Wiltshire Canada Agriculture and Agri-Food Canada Charlottetown Research and Development Centre Charlottetown Canada Fujian Agriculture and Forestry University Fuzhou China

Potato late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases in the agricultural sector around the world. Many genes (R genes) conferring resistance to late blight have been identified in various potato species and most of these R genes have been used in potato breeding. The aim of this study was to develop and validate PCR-based assays for the R genes Rpi-blb1, Rpi-blb2, Rpi-blb3 and Rpi-bt1, to distinguish between late blight resistant and late blight susceptible potato progeny in the given breeding background. A total of 100 breeding progeny were screened for the presence of these R genes and tested for resistance against P. infestans mating type A2, genotype US-8 strain, using detached leaf and tuber rot assays. PCR products for the Rpi-blb1 and Rpi-bt1 resistance genes were identified in the resistant progeny but were absent in the susceptible ones;therefore these PCR assays could differentiate between late blight resistant and susceptible plants. Genotypic data from the DNA markers derived from the Rpi-blb1 and Rpi-bt1 genes was found to correlate with the phenotypic data for foliar late blight but not with data for tuber rot. Our results demonstrate that markers derived from these two R genes could be useful for marker-assisted selection (MAS) for foliar late blight resistance in potato breeding programs.

关键词： Late Blight Phytophthora infestans Resistance R genes DNA Markers