蔬菜镉含量市场调查及对甜菜夜蛾生殖的影响

【作者】 杨勇；

【导师】 苏宏华； 杨进；

【作者基本信息】 扬州大学 ， 植物保护（专业学位）， 2019， 硕士

【摘要】 重金属污染日益严重,是世界上环境污染问题之一。重金属在食物链中极易传递和富集。重金属主要通过昆虫取食进入虫体,影响甜菜夜蛾的生长繁殖。但目前甜菜夜蛾在受到重金属胁迫后在昆虫体内的分布、对其生长发育以及生殖能力的影响及其影响机制还未见系统研究。本文系统研究了甜菜夜蛾四种常见寄主蔬菜中重金属的累积以及取食含不同镉浓度(0、0.2、3.2、51.2 mg/kg)的人工饲料后甜菜夜蛾体内重金属的分布,镉对其生长发育的影响,最后选取相关差异基因进行分析,主要研究结果如下:1甜菜夜蛾寄主蔬菜重金属含量分析采用原子荧光法检测甜菜夜蛾主要蔬菜寄主(青菜、葱、甘蓝以及芦笋)中铜、铅、镉、铬及汞五种重金属的含量。研究结果显示,4种寄主中汞未检出,铅、镉含量超标。对蔬菜的重金属富集能力分析显示,青菜与甘蓝中铅含量高,葱与芦笋中镉含量较高,甘蓝中铅含量最高,芦笋中铅、镉含量最高;从寄主重金属含量对比中,铜、铅含量青菜、葱及甘蓝最高,镉含量芦笋最高,铬含量甘蓝最高。2镉在甜菜夜蛾各组织中的分布本文研究镉胁迫后甜菜夜蛾体内不同组织所富集镉的含量,探讨幼虫、成虫、雌雄虫体内不同镉浓度(0.2、3.2、51.2 mg/kg)胁迫后各组织镉含量的差异。(1)5龄幼虫中肠的镉累积浓度最高,显著高于脂肪体,极显著高于表皮和血淋巴。(2)在各浓度中腹部的累积浓度显著高于头、胸、足、翅。对于雌雄个体间,相同浓度下,雌虫的头、胸、翅三个组织部位镉含量显著高于雄虫;雌雄虫的腹、足两组织镉含量无显著差异。(3)雄虫腹部组织在各浓度中脂肪体的累积浓度显著高于中肠、血淋巴、精巢;雌虫腹部组织在各浓度中脂肪体与卵巢的累积浓度显著高于中肠、血淋巴。相同镉浓度胁迫下雌虫卵巢的镉含量显著高于雄虫精巢;脂肪体、中肠与血淋巴中镉含量雌雄虫间无显著差异。3镉对甜菜夜蛾生殖的影响不同浓度镉(CK、0.2、3.2、51.2mg/kg)对于连续两代甜菜夜蛾生殖影响:(1)CK、0.2、3.2 mg/kg三种浓度下F1、F2代的存活率没有显著差异,51.2 mg/kg存活率显著低于前三个浓度且F2代显著低于F1代。(2)F1代、F2代的化蛹率随着镉浓度的升高而下降,3.2、51.2 mg/kg的F2代化蛹率与F1代化蛹率差异显著。(3)CK、0.2、3.2 mg/kg的F1代羽化率与F2代无显著差异,51.2 mg/kg的F1代羽化率与F2代差异极显著。(4)F1、F2代的产卵数随着浓度的升高而下降,3.2、51.2 mg/kg的F2代产卵数极显著的低于F1代。为了 了解镉胁迫对雌雄生殖影响的差异,试验将不同浓度胁迫下的成虫相互配对后,研究结果表明,母代雌虫受镉胁迫比雄虫受镉肋迫对产卵量的影响大。对仅雌虫受胁迫后的F2代进行连续胁迫发现:随着镉浓度的上升,F2代的存活率、化蛹率、羽化率和产卵量都下降,且除0.2 mg/kg浓度外,其余浓度各指标受胁迫的均小于不受胁迫的。4镉对甜菜夜蛾基因表达的影响不同镉胁迫对于甜菜夜蛾单雌产卵量有显著影响,并且影响卵巢在发育过程中的形态及超微结构,通过探究卵巢基因的表达可以了解镉胁迫的影响机制。本试验通过转录组测序技术,阐明四种不同镉胁迫下甜菜夜蛾卵巢差异表达基因,探究镉胁迫的影响甜菜夜蛾生殖的分子机制,从脂肪酸合成、氨基酸合成和激素合成通路中筛选相关基因进行实时荧光定量分析表达量进行验证。结果显示:随着浓度的增加脂肪酸合成酶两个相关基因的表达量呈上升趋势,乙酰辅酶A羧化酶两个相关基因的表达量随浓度的升高而下降,脂肪酸合成酶表达量在低浓度0.2mg/kg时最高随着浓度的下降表达量下降;随着浓度的增加酪氨酸蛋白激酶和天冬氨酸转氨酶相关基因的表达量不断上升,多酚氧化酶的表达量随浓度升高而下降;随着浓度的增加类蛋白、载脂蛋白和雌激素硫转移酶的表达量随浓度的升高而升高。更多还原

【Abstract】 Heavy metal pollution has been one of the environmental pollution problems.Heavy metals are easily transferred and enriched in food chain.The effects of heavy metal stress on the growth and reproduction of Spodoptera.exigua and its mechanism have not been systematically studied.The accumulation of heavy metals in four hosts of S.exigua and the accumulation and distribution of heavy metals in S.exigua with different cadmium concentrations(0,0.2,3.2,51.2 mg/kg)were systematically studied in this paper.Some indexes of growth and development were determined and the related differentially expressed genes were selected for analysis.The main results are as follows:1 Determination of heavy metal content in hosts of S.exiguaBecause heavy metals enter the body mainly through food intake,which affects the growth and reproduction of S.exigua.Therefore,five heavy metals(copper,lead,cadmium,chromium and mercury)in the main vegetable hosts of S.exigua(pakchoi,scallion,cabbage and asparagus)were determined.The results showed that mercury was not detected in four hosts,and lead and cadmium content exceeded the standard.It was found that pakchoi and cabbage had high accumulation ability of lead,the content of cadmium in scallion and asparagus was higher,the content of lead in cabbage was the highest,and the content of lead and cadminm in asparagus was the highest.Compared with host,the content of copper and lead in pakchoi,scallion and cabbage was the highest,the content of cadmium in asparagus was the highest,and the content of chromium in cabbage was the highest.2 Distribution of cadmium in tissues of S.exiguaThe cadmium contents in different tissues of S.exigua under cadmium stress were studied.The differences of cadmium contents in different tissues between larvae and adults under cadmium stress at different concentrations(0.2,3.2,51.2 mg/kg)and between males and females were investigated.(1)The cumulative concentration in the midgut of the 5th instar larvae was the highest,significantly higher than that in the fat body,and significantly higher than that in the epidermis and hemolymph.(2)For cadmium content in adult tissues,there were similar pattern between male and female adults at different concentrations,and the cumulative concentration of cadmium in abdomen was significantly higher than that in head,thorax,legs and wings.For males and females,the cadmium content in head,thorax and wings of females was significantly higher than that of males at the same concentration,while there was no significant difference between males and females in the cadmium content in abdomen and legs.(3)The accumulative concentration of cadmium In fat body of males was significantly higher than that in midgut,hemolymph and testis.3 Effect of cadmium stress on reproduction of S.exiguaThe effects of heavy metals on the growth and development of S.exigua and its offspring were studied.The reproductive effects of different concentrations of heavy metals(CK,0.2,3.2,51.2 mg/kg)on two generations of S.exigua were studied.(1)The survival rates of F1 and F2 generations with CK,0.2 and 3.2 mg/kg concentrations were not significantly different.The survival rates were 0.2>CK>3.2 mg/kg.Only 51.2 mg/kg survival rate was significantly lower than those of the other three treatments and the F2 generation was significantly lower than the F1 generation.(2)The pupation rate of F1 and F2 generations decreased with the increase of cadmium concentration.The pupation rate of F2 generation of 0.2 and 3.2 mg/kg was higher than that of F1 generation.The pupation rate of F2 generation of CK and 51.2 mg/kg was lower than that of F1 generation,and the pupation rate of F2 generation of 3.2 and 51.2 mg/kg was significantly different from that of F1 generation.(3)The emergence rate of CK and 0.2 mg/kg in F2 generation was slightly higher than that of F1 generation,while that of 3.2 and 51.2 mg/kg in F1 generation was lower than that of F2 generation,and there was no significant difference between CK,0.2 and 3.2 mg/kg in F1 generation and F2 generation.The emergence rate of 51.2 mg/kg in F1 generation was significantly different from that of F2 generation.(4)The number of eggs laid in F1 and F2 generations decreased with the increase of cadmium concentration.The number of eggs laid in F1 generation of CK and 0.2 mg/kg was slightly higher than that in F2 generation,and the number of eggs laid in F2 generation of 3.2 and 51.2 mg/kg was significantly lower than that in F1 generation.In order to understand the difference of reproductive effects between males and females,adults under different concentrations of cadmium stress were paired and found that:comparing the number of spawning after pairing on 0.2 mg/kg:only female was under stress>only male was under stress;on 3.2 mg/kg:only females were under stress<only males were under stress;on 51.2 mg/kg:only females were under stress<only males were under stress;In general,maternal females are more affected by cadmium stress than males by cadmium stress,which has a greater impact on oviposition.Subsequently,the continuous stress on female F2 generation showed that the survival rate,pupation rate,emergence rate and oviposition rate of F2 generation declined with the increase of the concentration,and the other concentration indexes were less than those of non-stressed F2 generation except 0.2 mg/kg concentration.4 Analysis of ovarian gene expression under cadmium stress and determination of related gene expressionDifferent cadmium stress had significant effects on the number of eggs laid by single female,and affected the morphology and ultrastructure of ovaries during development.The mechanism of cadmium stress could be understood by exploring the expression of ovarian genes.The transcriptional sequencing technique is to measure the sequence of the transcript by high throughput sequencing technology,so as to study the function and structure of the gene based on the whole level.In this experiment,the ovarian genomes of S.exigua under four different cadmium stresses were sequenced by transcriptional sequencing technique,and the mechanism of cadmium stress was explored from the gene level,and fatty acid synthesis was carried out from the point of view of gene level.The genes in Amino Acid Synthesis and hormone synthesis pathway were screened.The expression level was verified by Real time PCR.The results showed that the expression of two related genes of fatty acid synthesis increased with the increase of concentration,and the expression of two related genes of acetylcoenzyme A Carboxylase decreased with the increase of concentration.The expression of fatty acid synthase decreased with the decrease of concentration at low concentration of 0.2mg/kg.With the increase of concentration,the expression of tyrosine protein kinase and aspartate transaminase related genes increased,while the expression of polyphenol oxidase decreased with the increase of concentration.With the increase of concentration,the expression of apolipoproteins and estrogen thiosyltransferase increased with the increase of concentration.更多还原