Metabolomics - A robust bioanalytical approach for the discovery of the modes-of-action of pesticides: A review

The agrochemical industry is facing great undertaking that includes increasing demand for the development of new crop protection agents that are safe for the environment and the consumers, and at the same time combat the issue of the emergence of resistance pest strains. The mode-of-action (MoA) is among the features of a bioactive compound that largely determine whether the abovementioned issues are addressed or not, and subsequently whether its commercial development will be addressed. The early discovery of the MoA of bioactive compounds could accelerate pesticide research and development by reducing the required time and costs. Based on advances in synthetic and natural product chemistry, scientists have access to a vast number of compounds that could potentially be developed as crop protection agents. The screening of such compounds with respect to their MoA requires accurate and robust bioanalytical tools. Metabolomics is a powerful bioanalytical tool that will likely play a significant role in the acceleration of the discovery of MoA of bioactive compounds. In the present review, the capabilities and principles and applications of metabolomics for the study of the MoA of herbicides, insecticides, acaricides, fungicides, and antibiotics are discussed.     

Relationship between adipose maturity and fatty acid composition in various adipose tissues of Japanese Black,Holstein and Crossbred (F1) steers

The amount of monounsaturated fatty acid (MUFA) is intimately related to adipose softness, melting point (MP) and flavor in beef. Stearoyl‐CoA desaturase (SCD) is a main gene involved in MUFA synthesis. Mature adipose tends to be highly saturated, whereas immature or maturing adipose is highly unsaturated when chronologically based, so the degree of non‐saturation can be an index of adipose maturity. In this study, three different adipose tissues (coelomic (CL), perirenal (PR), and subcutaneous (SC)) from three beef breeds with differing slaughter ages (Japanese Black (29.5 months), Holstein (20.1 month), and F1 crossbreed (25.6 months)) were examined to: (i) determine adipose maturity level as indexed by MUFA %; and (ii) determine SCD and other lipogenic gene messenger RNA (mRNA) expression levels in relation to unsaturated fatty acid content. Fatty acid composition was significantly different between adipose tissues (P < 0.05). MUFA amount was high in the following order: SC > CL > PR. This pattern corresponded to SCD mRNA expression profile showing higher expression in SC than CL and PR. However, Japanese black cattle are an exception with CL adipose containing similar UFA % as SC adipose, yet having the lowest SCD mRNA expression level among all adipose tissues tested. Therefore, SCD mRNA expression and MUFA % appear to be directly related; however, differences in SCD mRNA expression among three adipose tissues may reflect differences in the fat development characteristics affected by chronological age of the cattle breeds.     

草莓八氢番茄红素脱氢酶基因pds的克隆及特征分析

 采用RT-PCR和RACE技术从草莓果实中克隆到草莓类胡萝卜素合成途径中关键基因pds,该cDNA全长2 043 bp,具有一个1 704 bp的完整开放阅读框（ORF）,编码568个氨基酸。序列分析表明,pds编码的氨基酸序列与其它植物的PDS蛋白有很高的相似性。系统进化树分析显示,草莓PDS与杏的PDS蛋白亲缘关系比较近。原核表达结果表明pds基因在大肠杆菌中获得高效表达。利用半定量RT-PCR技术进行组织表达模式分析发现,pds基因在草莓的花、叶片和果实中均有表达,表达量为红果＞粉红果＞白果＞花＞青果＞叶。     

鹤望兰八氢番茄红素脱氢酶基因SrPDS 的克隆及表达分析

 采用RT-PCR和RACE方法从鹤望兰黄色花萼中克隆到类胡萝卜素合成途径关键基因SrPDS, 该 cDNA 全长2 069 bp,具有完整的开放阅读框（ORF）,共1 746 个碱基,编码581 个氨基酸。氨基酸 同源性分析表明,SrPDS 推导的氨基酸序列与已报道的其他植物的PDS 蛋白具有很高的同源性。系统进 化树分析显示,鹤望兰SrPDS 与番红花首先聚为一类,其次与百合、水仙PDS 蛋白亲缘关系较近。应用 半定量PCR 分析表明,SrPDS 在黄色花萼和叶片中高表达,在蓝色花瓣和根中低表达;且在花发育的始 花期表达量最高,表明该基因在黄色花萼发育过程中起着重要作用。     

茶树八氢番茄红素脱氢酶cDNA全长克隆与表达分析

八氢番茄红素脱氢酶是类胡萝卜素生物合成途径的关键酶之一。本实验采用3’/5’RACE和RTPCR技术成功扩增出茶树八氢番茄红素脱氢酶基因（PDS）的3’端和5’端序列,序列拼接获得全长cDNA序列,命名为CsPDS,并将其登录至GenBank,登录号KF646537。经生物信息学分析,所得基因cDNA序列全长为2295 bp,开放阅读框（ORF）1749 bp,编码582个氨基酸,预测分子量约为64.86 kDa,理论等电点（PI）为6.77,属于亲水性蛋白。该基因编码的氨基酸序列与柿树PDS序列的同源性达到87%,多序列比对表明茶树PDS具有高度保守区域,基于邻接法的进化树显示与柿树、葡萄的亲缘关系最近。实时荧光定量PCR分析结果显示,CsPDS在‘黄金芽’体内表达没有受抑制,表明‘黄金芽’黄色白化可能不是在CsPDS基因转录水平异常而引起。     

T5代γ-亚麻酸转基因大豆的遗传稳定性分析

利用PCR检测和标记基因抗性检测对表达γ-亚麻酸转基因大豆第5代植株的外源基因遗传稳定性及目的基因和抗性基因的分离情况进行了分析。结果表明:在20个转基因大豆株系中,有2个转基因大豆株系(TS824和TS825)只含有△6-fad基因而没有bar基因;7个转基因大豆株系(TS81、TS83、TS85、TS87、TS811、TS814、TS818)只含有bar基因而没有△6-fad基因;9个转基因大豆株系(TS88、TS89、TS810、TS813、TS815、TS816、TS817、TS819和TS820)既有△6-fad基因也有bar基因;2个转基因大豆株系(TS82和TS86)既没有△6-fad基因也没有bar基因。因此,△6-fad基因在部分株系中获得了稳定的遗传,同时有2个株系目的基因和抗性基因在后代中分离。     

花生Δ9-硬脂酰-ACP脱氢酶基因(SAD)的序列分析

利用同源克隆技术获得花生区组二倍体野生种A. duranensis和A. ipaensis Δ⁹-硬脂酰-ACP脱氢酶基因SAD (命名为gSAD-A和gSAD-B)及3个栽培品种的SAD,每个栽培品种有2个SAD (命名为gSAD-1和gSAD-2)。同时获得丰花2号SAD的两条全长cDNA (命名为FhrSAD-1和FhrSAD-2)。丰花2号的FhgSAD-1和FhgSAD-2均含有2个内含子,二者同源性97.5%,共有69个变异位点,其中62个是SNP位点、6个特异性酶切位点。FhrSAD-1和FhrSAD-2间核苷酸序列同源性98.6%,其中编码区序列同源性98.9%,共有12个变异位点,编码的Ah-SAD2氨基酸序列与Ah-SAD1相比在N端的¹⁷PSSSSSSSSSSFSL³⁰丝氨酸聚集区少一个丝氨酸。gSAD-1和gSAD-A同源性为99.9%,存在4个SNP位点; gSAD-2和gSAD-B同源性为100%。推测gSAD-1和gSAD-2分别来自花生栽培品种的A、B2个染色体组。研究明确了花生不同染色体组SAD的序列特征,为进一步探讨SAD的表达及其在控制花生籽仁脂肪酸组分中的作用提供了重要的参考。     

花生油酸脱氢酶基因遗传转化体系的建立与表达分析

 【目的】培育高油酸的花生新种质。【方法】以根癌农杆菌为介导将含有油酸脱氢酶基因的植物双元表达载体pFGC/2nd转入花生外植体丛生芽,通过卡那霉素筛选,器官发生再生转化植株;荧光定量PCR检测转基因植株。【结果】丛生芽外植体于OD600为0.6的农杆菌菌液中浸泡8～10 min后,在MS培养基上暗培养3 d效果较好。诱导筛选培养3个月左右,共得到16株转化植株,其中11个转基因植株经PCR检测呈阳性,说明外源基因已整合进入受体植物。进一步的荧光定量PCR检测显示,有4个植株基本上不表达或表达量很低。【结论】建立花生丛生芽遗传转化体系;RNA干扰对内源的油酸脱氢酶基因产生了抑制作用。     

高通量检测花生油酸含量相关基因AhFAD2等位变异的方法

花生(Arachis hypogaea)△12脂肪酸去饱和酶基因(△12 fatty acid desaturase,AhFAD2A和AhFAD2B)是决定花生籽粒油酸含量和高油酸亚油酸比值(oleic acid/linoleic acid,O/L)的关键基因,高油酸花生品种中这2个基因均发生突变.针对普通油酸花生和高油酸花生AhFAD2A 448位G/A差异和AhFAD2B 442位A碱基的插入/缺失(insertion-deletion,InDels)等位差异,已开发了包括酶切扩增多态性序列法(cleaved amplified polymorphic sequence,CAPS)和等位基因特异PCR(allele-specific PCR,AS-PCR)等多种检测的标记和检测方法.本研究针对上述2个SNP位点,开发了可进行高通量检测的实时定量PCR引物、TaqMan 探针和检测技术,该方法检测效率和准确率均很高.本研究还开发了更为经济和高效的竞争性等位基因特异性PCR(kompetitive allele specific PCR,KASP)引物和检测方法.利用开发的TaqMan探针检测方法和KASP方法检测了14个花生品种和高油酸选育回交组合BC2F1和BC1F2群体部分种子的基因型,并比较了两种方法检测结果的一致率,发现这两种方法检测AhFAD2B 442 nt A/-InDel差异结果的一致率高达96.7％;而针对AhFAD2A 448位G/A差异位点检测一致率仅为71.7％.本研究还比较了目前常用的CAPS、AS-PCR、测序法、TaqMan探针法及KASP方法的优缺点,对相关方法的应用前景进行了讨论.本研究涉及的高通量检测方法可有效地辅助高油酸品种的选育,极大地提高了目标性状选择的准确性和效率.