我校首次获准国家自然科学基金立项

由我校特聘教授，留学加拿大、美国的归国博士张建社领导的生物工程与环境科学系科研小组申请的国家自然科学基金课题——“名贵鱼类肉质性状的蛋白分子基础及相关基因克隆”（批准号：30571414），成功获准立项，实现了我校去年升本以来国家级自然科学基金零的突破。本项目拟采用生物化学，蛋白质组学以及分子生物学的技术和方法，从名贵鱼类中分离与肉质相关的蛋白，而后进行氨基酸组成成份分析，筛选高含量的与人类营养健康相关的氨基酸和蛋白质，继而筛选优良肉质性状相关基因，并进行相关基因表达调控和鱼的转基因研究。     

肉质性状相关基因在四种鲤科鱼类肌肉组织中的表达差异研究

为了阐明肉质性状相关基因在四种鲤科鱼类中的表达差异,以齐口裂腹鱼(雅鱼,Schizothorax prenanti)、草鱼(Ctenopharyngodon idellus)、鲤鱼(Cyprinus carpio)和鲢鱼(Hypophthalmichthys molitrix)四种鲤科鱼类为研究对象,利用索式抽提法和物性测定仪测定四种鱼类背部与腹部肌内脂肪(IMF)含量和肌肉剪切力,并利用荧光定量PCR(q PCR)检测心脏型脂肪酸结合蛋白(H-FABP)、过氧化物酶体激活增殖受体γ(PPARγ)、过氧化物酶增殖剂受体γ亚型的辅激活因子1α(PGC-1α)、钙蛋白酶抑制蛋白(CAST)等肉质性状相关基因在四种鱼肌肉组织中的表达.结果显示,齐口裂腹鱼背部与腹部肌肉的IMF含量极显著高于其他鱼类(P<0.01),而肌肉剪切力均显著低于其他三种鱼类(P<0.05),PGC-1α在齐口裂腹鱼背部及腹部肌肉中表达水平最高,PPARγ和CAST在齐口裂腹鱼背部及腹部肌肉中表达水平最低.研究结果为阐明齐口裂腹鱼优良肉质形成的分子机制提供重要的参考数据.     

鱼类生长激素基因工程研究

生长激素是鱼体生长发育最重要的内分泌激素.20世纪30年代以来,在对生长激素生物化学功能研究的基础上,对鱼类生长激素的生理功能和作用机制进行了系统深入的探讨,为鱼类生长激素基因工程研究奠定了基础.随着分子生物学技术的发展,40多种鱼类的生长激素基因及其cDNA被克隆,鱼类生长激素基因工程研究取得了一系列重要进展:应用DNA重组和基因转移技术,获得了多种转生长激素基因鱼,由于重组生长激素基因的"内源性"表达,转基因鱼获得生长快、饵料转化效率高等优良生产性状,显示了生长激素基因在鱼类基因工程育种中的广阔应用前景;另一方面,鱼类生长激素基因在工程菌中表达的技术体系得以建立和发展,使获得大量廉价的鱼类生长激素产品成为可能,为渔业养殖新型饵料添加剂的研制开辟了新的途径.     

一个优良养殖对象——宜宾鲴

我省江河纵横,水域广阔,鱼类资源丰富,蕴藏着许多有发展前途的经济鱼类.江河鱼类资源调查的任务之一,是为内塘、溪河养殖寻找新的品种,研究其生物学,为移养驯化提供科学依据.在嘉陵江鱼类资源调查中,根据各项生物学指标的分析研究,我们认为,我省特有的宜宾鲴,是一种优良的养殖对象.它具有适应性强,生长较快,性成熟早,肉质肥嫩的特点.以藻类和植物碎屑为食,不与“家鱼”争夺饵料,且能起到充分利用水体.充分利用饵料,又可高密度混养的增产效果.因此,作为在溪河、塘堰、水库中的移养驯化对象,是值得宣传推广的.这里对宜宾鲴的生物学性状作一简要介绍.     

建鲤品种特性研究进展

建鲤是遗传性状比较稳定的人工育成的鱼类品种,生长快,体型体色美观,肉质好,适应性和抗病力强,生产性能优良.深入研究建鲤的形态特征、经济性状和遗传性能,保持建鲤优良的遗传特性,发挥其品种优势,扩大生产应用规模.     

鳜鱼(S.Keneri)肌球蛋白重链基因cDNA的克隆及其特征分析

鳜鱼(S.Keneri)以优良肉质性状成为极具商品价值和大力推广人工养殖名贵鱼类之一.为掌握控制优良肉质性状的遗传基础,采用同源克隆RT-PCR方法,克隆出该鱼肌球蛋白重链(MYH)cDNA序列.该基因cDNA序列全长5938bp,编码区长度为5814bp,含有poly(A)信号,3’非翻译区长124bp,其开放阅读框共编码1938个氨基酸,推算蛋白质分子质量为221.6Ku.鳜MYH基因由3个结构域组成,即MYSc-type-Ⅱ,SMC-N和Myosintail 1,其中MYSc-type-Ⅱ头部结构与鲤、斑马鱼同源性达90%,具高度保守性.通过DNAstar软件MegAlign构建进化树显示不同物种间该基因核苷酸编码序列同源性为77%—86%,氨基酸编码序列同源性达80%—92%.采用RT-PCR方法研究MYH在不同发育阶段差异表达结果表明,MYH在肌肉效应期开始有低量表达,成鱼期和鱼苗期表达量高,而囊胚期与神经胚均未表达.由此推测MYH基因于肌肉效应期开始表达.研究结果揭示鳜鱼肌球蛋白重链基因在进化上保持与其他脊椎动物相似性和差异性,为决定名贵鱼类肉质结构基因的研究提供了分子生物学基础.     

牛肉质性状功能基因组学研究进展

功能基因组学的研究使人们能够从分子水平上对牛肉品质进行改良,从而得到优质高档的牛肉.牛的肉质性状与牛肉的经济价值紧密相关,其主要包括嫩度、大理石花纹、多汁性等.鉴于近年来牛肉质性状相关领域的研究比较活跃,利用68篇文献对牛的主要肉质性状的QTL、SNP遗传标记、cDNA文库以及与牛肉质性状相关的基因的研究现状和进展进行了综述.     

畜禽养殖废水中抗生素和抗性基因的去除技术进展

近年来抗生素在养殖业中大量使用，致使畜禽养殖废水成为抗生素及其抗性基因的重要储库，成为环境中抗生素及其抗性基因的重要来源之一.在分析养殖废水中抗生素及其抗性基因含量的基础上，介绍可同时去除畜禽养殖废水中抗生素及其抗性基因的处理技术.在分析各处理方法的优缺点的基础上，提出一种可行的畜禽养殖废水抗生素及其抗性基因处理技术.     

鳜鱼（S．Keneri）肌球蛋白重链基因cDNA的克隆及其特征分析

鳜鱼（S．Keneri）以优良肉质性状成为极具商品价值和大力推广人工养殖名贵鱼类之一．为掌握控制优良肉质性状的遗传基础,采用同源克隆RT-PCR方法,克隆出该鱼肌球蛋白重链（MYH）cDNA序列．该基因cDNA序列全长5938bp,编码区长度为5814bp,含有poly（A）信号,3’非翻译区长124bp,其开放阅读框共编码1938个氨基酸,推算蛋白质分子质量为221．6Ku．鳜MYH基因由3个结构域组成,即MYSc-type-Ⅱ,SMC-N和Myosintail1,其中MYSc-type-Ⅱ头部结构与鲤、斑马鱼同源性达90％,具高度保守性．通过DNAstar软件MegAlign构建进化树显示不同物种间该基因核苷酸编码序列同源性为77％86％,氨基酸编码序列同源性达80％-92％．采用RT-PCR方法研究MYH在不同发育阶段差异表达结果表明,MYH在肌肉效应期开始有低量表达,成鱼期和鱼苗期表达量高,而囊胚期与神经胚均未表达．由此推测MYH基因于肌肉效应期开始表达．研究结果揭示鳜鱼肌球蛋白重链基因在进化上保持与其他脊椎动物相似性和差异性,为决定名贵鱼类肉质结构基因的研究提供了分子生物学基础．     

金华猪优良肉质性状关键功能基因挖掘及营养调控机制

正猪肉是老百姓餐桌上必不可少的肉食品,虽然国内地方品种猪的肉质性状较为优良,但生长速度跟不上需求,因此生产速度快、饲养效益高的国外品种猪成为养殖户们的首选。浙江大学汪以真团队的"金华猪优良肉质性状关键功能基因挖掘及营养调控机制"项目,通过挖掘地方猪金华两头乌的优良肉质性状关键功能基因,解析了营养靶向调控机制并形成了一套改善猪肉品质的饲料营养关键技术,为改善猪肉品质和优质猪肉生产提供了调控新策略。该成果荣获2020年度浙江省自然科学奖一等奖。     

基于SVM-RFE的水稻抗病基因筛选

提出一种改进的回归特征消去支持向量机特征选择方法(SVM-RFE)对水稻的抗病基因进行筛选. 实验结果表明: 在预测得到的20个与水稻抗病/敏感相关基因中, 有3个基因与已知的水稻抗病基因紧密相关; 2个基因与已知的水稻抗病基因有一定的相关性. 通过该方法能找到影响水稻生长状态（正常/染病）的基因.     

鱼类饲料甘草添加剂的研究

研究了甘草作为鱼类新型饲料的抗病功效试验；甘草抗鱼病有效成分的提取及其定性定量分析以及白头公司、远志、陈皮的抗病有效成分分析及其抗鱼类病对照试验。试验期间，甘草提取的与饵料配合供养殖鱼摄食，有使于类提高抗病性、保持健康的功效。     

一株生防烟管菌几丁质酶表达及抗真菌活性

为了探究烟管菌BK-1的生物防治潜力,本研究测试了一株具有生防菌活性的烟管菌BK-1的抗病效果,并使用RT-qPCR分析了该菌株的8个细胞壁裂解酶基因在与不同病原真菌对峙培养中的表达水平,结果表明,在应对链格孢、茄链格孢、尖孢镰刀菌和灰葡萄孢这4种病原真菌时,几丁质酶基因BaCHIB表达上调了27.3至50.3倍,远高于其他7个基因.BaCHIB属于糖苷水解酶18家族(GH 18),并具有Chic_BD结构域,属于B类几丁质酶(CHIB).结合基因表达谱分析,该基因诱导表达上调情况的出现与抗病效果的出现高度同步.外源表达蛋白BaCHIB显示出较高的几丁质酶活性,且表现出对多种植物病原真菌,包括链格孢,链格孢和灰葡萄孢显著的抗真菌活性,显示其对部分病原真菌具有拮抗能力.     

Gene expression in Pseudomonas aeruginosa biofilms.

Bacteria often adopt a sessile biofilm lifestyle that is resistant to antimicrobial treatment. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections. To gain insights into the differences between free-living P. aeruginosa cells and those in biofilms, and into the mechanisms underlying the resistance of biofilms to antibiotics, we used DNA microarrays. Here we show that, despite the striking differences in lifestyles, only about 1% of genes showed differential expression in the two growth modes; about 0.5% of genes were activated and about 0.5% were repressed in biofilms. Some of the regulated genes are known to affect antibiotic sensitivity of free-living P. aeruginosa. Exposure of biofilms to high levels of the antibiotic tobramycin caused differential expression of 20 genes. We propose that this response is critical for the development of biofilm resistance to tobramycin. Our results show that gene expression in biofilm cells is similar to that in free-living cells but there are a small number of significant differences. Our identification of biofilm-regulated genes points to mechanisms of biofilm resistance to antibiotics.     

Analysis of porcine MHC expression profile

The porcine major histocompatibility complex (MHC, also named swine leukocyte antigen, SLA) is associated not only with immune responsibility and disease susceptibility, but also with some reproductive and productive traits such as growth rate and carcass composition. As yet systematical research on SLA expression profile is not reported. In order to illustrate SLA expression comprehensively and deepen our understanding of its function, we outlined the expression profile of SLA in 51 tissues of Landrace by analyzing a large amount of ESTs produced by ““Sino-Danish Porcine Genome Project““. In addition, we also compared the expression profile of SLA in several tissues from different development stages and from another breed (Erhualian). The result shows: (i) classical SLA genes are highly expressed in immune tissues and middle part of intestine; (ii) although SLA-3 is an SLA la gene, its expression abundance and pattern are quite different from those of the other two SLA la genes. The same phenomenon is seen in HLA-C expression, suggesting that the two genes may function similarly and undergo convergent evolution; (iii) except in jejunum, the antigen presenting genes are more highly expressed in breed Erhualian than in Landrace. The difference might associate with the higher resistance to bad conditions (including pathogens) of Erhualian and higher growth rates of Landrace.     

海洋鱼类的生长与发育生物学研究与苗种培育

鱼类生长和发育生物学是海洋鱼类苗种培育的理论基础。近年来鱼类生长与发育生物学取得的研究进展为改善海洋鱼类苗种培育提供依据。主要的研究进展是：阐明调控鱼类生长的脑－脑垂体－肝脏轴的种激素分泌活动和作用机理,促进海洋鱼类生长激素释放和提高生长率;海洋鱼类幼苗生长发育的形态学与生理学特征及对饵料的消化吸收;海洋鱼类幼苗生长发育所需要的营养物质;促进鱼类苗种对饵料摄取与消化吸收的饵料添加剂;环境条件对海洋鱼类苗种利用营养物质和生长的影响等。目前所取得的进展还只限于少数几种海洋养殖鱼类。许多基本理论问题尚未充分阐明。今后,为配合海洋鱼类养殖和增殖的发展,必需系统深入研究各种重要海洋养殖鱼类的生长与发育生物学,并在此基础上建立苗种培育新技术。     

Gene expression profiling related to powdery mildew resistance in wheat with the method of suppression subtractive hybridization

“Bainong 3217 × Mardler” BC₅F₄ wheat line at the initial stage of inoculation with powdery mildew pathogen (Erysiphe graminis DC) was used to construct a suppression subtractive hybridization (SSH) cDNA library. Totally 760 ESTs were obtained through sequencing. Similarity analysis of ESTs based on BLASTn and BLASTx with the sequences in GenBank, in combination with macroarray differential screening, revealed that 199 ESTs of 65 kinds were known to be functionally disease resistance related. Based on the gene expression profiling in the present study, it is postulated that salicylic acid (SA) and MAP-related signal transduction pathways were involved in powdery mildew resistance in wheat. System acquired resistance genes were predominant in terms of kinds and quantity. With the initiation of cell defense reaction, the genes conferring anti-oxidation substances were largely expressed and thus cell protection mechanism was activated. Much evidence revealed that phenylpropanes metabolic pathway was involved in phytoalexin synthesis in wheat powdery mildew resistance. Genes conferring some enzymes of structural modification of cell walls and proteinase inhibitors inhibiting pathogen growth were also detected. The genes controlling a few proteinases (mainly cysteine proteinase) had a considerable redundancy of expression.     

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.     

Hedgehog signalling controls eye degeneration in blind cavefish

Hedgehog (Hh) proteins are responsible for critical signalling events during development but their evolutionary roles remain to be determined. Here we show that hh gene expression at the embryonic midline controls eye degeneration in blind cavefish. We use the teleost Astyanax mexicanus, a single species with an eyed surface-dwelling form (surface fish) and many blind cave forms (cavefish), to study the evolution of eye degeneration. Small eye primordia are formed during cavefish embryogenesis, which later arrest in development, degenerate and sink into the orbits. Eye degeneration is caused by apoptosis of the embryonic lens, and transplanting a surface fish embryonic lens into a cavefish optic cup can restore a complete eye. Here we show that sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh) gene expression is expanded along the anterior embryonic midline in several different cavefish populations. The expansion of hh signalling results in hyperactivation of downstream genes, lens apoptosis and arrested eye growth and development. These features can be mimicked in surface fish by twhh and/or shh overexpression, supporting the role of hh signalling in the evolution of cavefish eye regression.