甘蓝型油菜烷羟化酶基因MAH1的克隆与表达分析

在植物蜡质合成途径中,中链烷烃羟化酶(mid-chain alkane hydroxylase,MAH)催化烷烃羟基化形成二级醇,进一步氧化为酮。本研究以拟南芥P450依赖性酶CYP96A15/MAH1基因为探针,采用电子克隆与RT-PCR技术,获得2个甘蓝型油菜MAH1的全长编码区序列,分别命名为BnMAH1-1和BnMAH1-2(Gen Bank登录号分别为KT795344和KT795345)。二者ORF长度均为1491 bp,无内含子,核苷酸与氨基酸序列分别有92.4%与90.9%的一致性。根据编码区预测的BnMAH1-1和BnMAH1-2前体蛋白均为包含496个氨基酸残基的多肽链,具有典型的P450蛋白家族保守结构P415x R417x、K螺旋(E359xx R362)、C末端的血红素结合域(F436xx Gx Rx Cx G445)以及氧结合带保守区域(A/G)G309x(D/E)T312(T/S)。NCBI Blast N、氨基酸序列多重比对与系统学分析表明,两者与拟南芥MAH1/CYP96A15同源性最高。实时荧光定量PCR表明,BnMAH1-1与BnMAH1-2主要在甘蓝型油菜茎、叶、花、及角果中表达,其中在叶片中的表达量最高,在根系中的表达量很低,这与角质层蜡质主要沉积在植株地上部分相一致。BnMAH1-1和BnMAH1-2在无蜡粉材料茎、叶片中几乎不表达,表明蜡质的减少与MAH1的转录下调有关。BnMAH1-1与BnMAH1-2受SA、Me JA、ACC、ABA、Na Cl及干旱胁迫诱导表达,其中BnMAH1-1可能在水分胁迫响应中起主要作用。     

Attachment and adhesion of conidia of Stagonospora nodorum to natural and artificial surfaces

Attachment and adhesion of conidia of a wheat-isolate of Stagonospora nodorum to leaf and artificial surfaces was studied. Attachment of conidia was a non-viable process, separate from adhesion, that occurred rapidly and irreversibly. Attachment involved conidial-surface carbohydrates and was partially influenced by surface hydrophobicity. The subsequent adhesion, via the secretion of extracellular matrix from conidia, was a viable process that induced the complete cover of conidia in response to wheat leaf surface components containing epi-cuticular wax and to a lesser extent to barley but inducing only partial covering on glass. Results suggest that specific surface components from the compatible host promote rapid attachment and adhesion of S. nodorum conidia.     

节节麦不同生育期叶片蜡质组成和超微形态的变化

为了解节节麦(Aegilops tauschii Coss.)不同生育期叶片表皮蜡质组成及晶体形态的变化,对节节麦叶片蜡质进行提取,利用气相色谱-质谱(GC-MS)联用仪和气相色谱-火焰离子化检测仪(GC-FID)对蜡质进行了定性和定量分析,并采用扫描电镜(SEM)对叶片表面蜡质晶体形态进行了观察。结果表明,节节麦叶表皮蜡质经GC-MS分析,共鉴定出化合物21种,主要以初级醇为主(苗期、抽穗期和灌浆期相对含量分别为85%、84%和70%),并含有少量的烷烃、醛、酮和脂肪酸;随着节节麦的生长发育,蜡质总量不断积累,烷烃含量极显著地升高,其中以C29烷烃的增加最明显,而初级醇含量显著降低,其中C26醇的相对含量降低最明显(苗期、抽穗期和灌浆期相对含量分别为80%、76%和63%),其他各类化合物组分变化不大。经扫描电镜观察,节节麦叶片近轴和远轴面的蜡质晶体形态无明显差别,苗期均为片状,抽穗期大部分仍以片状形态存在,并且蜡质晶体的密集程度比苗期稀疏,灌浆期蜡质晶体的密集程度变得更加稀疏。这些变化可能与抽穗期、灌浆期烷烃含量的增加和初级醇含量的减少有关。     

单宁-醛-胶原的反应——对植醛结合鞣机理的再认识

以黑荆树单宁为植物鞣剂，嗯唑烷为醛鞣剂，较系统地研究了氢键、疏水键、胶原氨基在植鞣、醛鞣和植-醛结合鞣中的作用。证实了植-醛结合鞣的反应机理与已有的认识有很大的区别。醛类化合物即使在植鞣后加入，其主要的反应基团仍然是皮胶原的氨基。但是当单宁分子中含有亲核活性较高的反应基团时，醛类化合物可以在皮胶原和单宁分子之间形成交联键。这种交联作用可以对提高皮胶原的热稳定性产生协同效应，而且使单宁与皮胶原的结合稳定性进一步加强，能够抵御氢键破坏试剂和疏水键破坏试剂的作用。采用缩合类单宁时，容易形成胶原-醛-单宁交联键；但采用水解类单宁时，形成这种交联键的几率很小。     

Chemical composition and antifungal activity of cuticular wax isolated from Asian pear fruit (cv. Pingguoli)

In order to understand the cuticular wax properties of Asian pear fruits (cv. Pingguoli), a series of assays was conducted on extraction, fractionation and determination of composition. The results showed that the maximum yield of wax from the fruit was obtained by chloroform/methanol (3:1), and then by chloroform extraction. Polar and low polar fractions occupied 64.3% and 26.9% of total crude wax by column chromatography. 54 and 22 components were identified in polar and low polar fractions by GC/MS, respectively. Long-chain fatty acids and triterpenoids predominated in polar fraction and n-alkanes were predominant in low polar fraction. In vitro tests indicated that polar fraction, low polar fraction and total crude wax inhibited spore germination and mycelial growth of Alternaria alternata, which is a causal agent of Alternaria rot of pears, and the maximum inhibition was obtained by polar fraction. These findings indicated that n-alkanes in low polar fraction and triterpenoids, fatty acid in polar fraction might have contributed to the antifungal properties of pear fruit cuticular waxes.     

鹅绒皮加工过程中组织结构变化的研究Ⅲ．鞣制后鹅皮肤及被毛的亚微结构

取经过浸酸处理（浸酸液：硫酸３ｇ／Ｌ，甲酸１ｇ／Ｌ，羟基乙酸１ｇ／Ｌ，芒硝６０ｇ／Ｌ，食盐３０ｇ／Ｌ）的冬鹅皮，分别用４种不同方法鞣制后，用透射电镜和扫描电镜观察了其亚微结构变化。结果表明，用由戊二醛（３ｇ／Ｌ）、三氧化二铬（０．６ｇ／Ｌ）、食盐（４０ｇ／Ｌ）、芒硝（４０ｇ／Ｌ）组成的鞣制剂鞣制，效果最佳。用其鞣制后，鹅皮肤的胶原纤维交联度增加，交联键增多，织角增大，原纤维增粗，纤维结合较紧密；鹅被毛仅羽小枝变化较明显，其顶端由正常的细尖状变为弯曲状。     

家蚕表皮蛋白基因的生物信息学分析

家蚕的表皮蛋白(cuticular protein)是家蚕重要的结构蛋白,与几丁质一同作为家蚕表皮的重要组成部分。运用生物信息学的方法对家蚕表皮蛋白进行了广泛分析。通过保守的基序检索家蚕基因组,得到163条候选的表皮蛋白序列,包括RR、Tweedle、CPF&CPFL等家族,并且发现具有确定的保守基序的表皮蛋白基因在染色体上都是以串联集群形式分布。氨基酸组成分析表明,这些表皮蛋白富含甘氨酸、丙氨酸、亮氨酸、组氨酸等。运用Sig-nalP server预测这部分蛋白质有85%都具有信号肽。进化分析显示,负选择是家蚕表皮蛋白基因进化的主要驱动力。采用TFSEARCH等在线服务软件分析发现,在70%含有RR基序的家蚕表皮蛋白基因的核心启动子区具有通用的TATAAA序列。     

大麦表皮蜡质的组分及晶体结构分析

为了解大麦不同生长发育时期各器官蜡质的组成及其晶体结构,以大麦品种Morex 为材料,利用气相色谱（GC）技术对其叶片、穗下茎和叶鞘三个器官的表皮蜡质成分进行了分析,并对其蜡质晶体结构进行扫描电镜观察。结果表明,从大麦品种Morex 表皮蜡质中鉴定出烷烃、初级醇、醛、脂肪酸和二酮等20种主要化合物成分,且蜡质碳链长度的分布范围主要为C₂₂~C₃₃。大麦叶片中初级醇含量最高,以C₂₆ 醇为主,其次是烷烃、醛和二酮,脂肪酸最少。大麦孕穗和扬花期,不同器官间蜡质总含量有显著差异,叶鞘最高,穗下茎最少。二酮在不同器官中都存在,其中,叶鞘和穗下茎中β-二酮的含量最高。扫描电镜观察表明,叶片表皮蜡质晶体结构均为片状,而叶鞘和穗下茎表皮晶体蜡质结构均为棒状。     

Cuticular penetration of β-cypermethrin in insecticide-susceptible and resistant strains of Bactrocera dorsalis

Semi-thin sectioning and transmission electron microscope techniques were employed to investigate the cuticle thickness, integument structure, and fat body of larvae from susceptible and resistant strains of Bactrocera dorsalis. The results showed that the cuticle of β-cypermethrin-resistant strains (25.96 ± 1.00 μm) was thicker than that of susceptible strains (19.36 ± 0.82 μm). The number of chitin layers in the endocuticle of β-cypermethrin-resistant strains (98.00 ± 3.61 layers) was more than that in susceptible strains (75.67 ± 2.40 layers). Compared with susceptible strains, the laminated structure of the chitin layers in the endocuticle of resistant strains revealed higher density and more distinctive structure, and the interspace of epidermal cells was thicker. Fat body in the resistant insects contained more fat granules than those in susceptible insects. Moreover, HPLC analysis showed that the cuticular penetration of β-cypermethrin into larvae of resistant strains was slower than that of susceptible strains. In addition, the metabolism of β-cypermethrin in resistant strains was faster than that in susceptible strains, indicating that the resistant strains could enhance detoxification metabolism. These results indicated that cuticle thickness, fat body, laminated structure of the chitin layers, and interspace of epidermal cells might be correlated with cuticular penetration between susceptible and resistant strains, suggesting that the resistant strains could decrease the rate of penetration of insecticide into the internal cavity.     

The clone of laccase gene and its potential function in cuticular penetration resistance of Culex pipiens pallens to fenvalerate

Decreased insecticides cuticular penetration, as one of resistant mechanisms in insect, has been extensively documented. Laccases, are enzymes with p-diphenol oxidase activity, was related to the cuticular tanning in insect. In this study, one laccase 2 gene (CpLac2) was cloned from Culex pipiens pallens. The CpLac2 contains an open reading frame (ORF) of 2289 bp and encodes a putative 762 amino acid protein. The deduced protein of CpLac2 was more similar to laccase 2 than other insect laccases, and shared the highest identity with laccases from the same family mosquito, Aedes aegypti and Anopheles gambiae. The developmental expression model of CpLac2 in C. pipiens pallens was measured by RT-PCR. The result showed the CpLaC2 was abundantly expressed in egg, the 4th instar larva and pupa, which suggested the role of CpLac2 for egg chorion tanning and cuticular sclerotization. Meanwhile, the expression of CpLac2 in fenvalerate-susceptible and -resistant strains of C. pipiens pallens was measured by real-time PCR. The result revealed the CpLac2 was significant higher expressed in resistant strain than in susceptible strain. The overexpression of CpLac2 in resistant strain suggested that resistance could derive from reinforcement of the cuticle, which decreased the penetration of insecticide in cuticle.