蚕卵指数孵化率的影响及孵化相关基因表达分析
作者:王雪珍 徐娇 王闪闪 浦月霞 沈兴家 唐顺明
来源:《南方农业学报》2020年第12期
        摘要:【目的】分析家蚕卵形指数对孵化率的影响规律,以及孵化酶基因(BmHE I和BmHE II )、几丁质酶基因(Chitinase)和周期蛋白基因(Period)在不同卵形间的表达规律,为阐明家蚕卵形指数与孵化率的相关性及其分子机理打下基础。【方法】调查家蚕Nistari品种野生型正常形品种(Nistari+)及其新突变体纺锤形品种(Nistari-)的卵形指数、孵化率、受精率和转青死卵率,分析其卵形指数与孵化率的相关性;并通过实时荧光定量PCR检测蚕卵胚胎发育后期孵化相关基因的表达情况。【结果】Nistari+蚕卵的平均卵形指数为1.26,极显著低于Nistari-蚕卵(1.73)(P<0.01,下同);孵化率为96.22%,极显著高于Nistari-蚕卵(51.33%)。当Nistari+蚕卵的卵形指数为1.24~1.25时,其孵化率(9
7.39%)和受精率(98.84%)均最高,转青死卵率最低(2.61%);而Nistari-蚕卵的卵形指数为1.77~1.81时,其孵化率(56.52%)和受精率(74.87%)均最高,转青死卵率最低(43.48%)。相关分析结果表明,Nistari蚕卵孵化率、受精率和转青死卵率与卵形指数存在一定的相关性,且受卵形指数影响。在蚕卵胚胎发育后期(孵化前),Nistari+蚕卵的BmHE I、BmHE II、Chitinase和Period基因大量表达,对应的相对表达量显著(P<0.05)或极显著高于NISTAR-蚕卵。【结论】家蚕Nistari品种的卵形指数与孵化率存在一定相关性,而BmHE I、BmHE II、Chitinase和Period基因的表达差异是导致Nistari-蚕卵与Nistari+蚕卵孵化率差异的分子基础。
        关键词: 家蚕;Nistari品种;卵形指数;孵化率;孵化相关基因
        中图分类号: S882.4 文献标志码: A 文章编号:2095-1191(2020)12-3109-07
        Abstract:【Objective】The effects of silkworm egg shape index (ESI) on hatching rate(HR), and the expression patterns of hatching enzyme genes(BmHE I and BmHE II), chitinase gene(Chitinase) and period protein gene(Period ) in different egg shapes were investigated, which laid a foundation for elucidating the correl
ation between egg shape index and hatching rate of silkworm eggs and its molecular mechanism. 【Method】The ESI, HR, fertilization rate(FR), and mortality rate in egg with pigmentary body of embryo(MR) were investigated between the wild type normal egg variety(Nistari+)and its new mutant spindle egg variety (Nistari-), and the correlation of ESI with HR were analyzed. And investigated the differences in gene expression levels related to hatching process in the later stages of embryonic development by real-time quantitative PCR. 【Result】The mean ESI of Nistari+ was 1.26, which was extremely lower than that of Nistari-(1.73)(P<0.01,the same below); the HR of Nistari+was 96.22%, which was extremely higher than that of Nistari- silkworm eggs(51.33%). When the ESI of Nistari+ was 1.24-1.25, the HR (97.39%) and FR(98.84%) were the highest and the MR(2.61%) was the lowest, while when the ESI of Nistari- was 1.77-1.81, the HR(56.52%) and FR(74.87%) of its eggs were the highest, MR was the lowest(43.48%). The results of correlation analysis showed that there was a certain correlation between the Nistari ESI and HR, FR and MR, and was affected by ESI. In the later stages of silkworm egg em
bryo development(before hatching), the expression levels of BmHE I, BmHE II, Chitinase and Period genes of Nistari+ silkworm eggs were high, and were significantly(P<0.05) or extremely(P<0.01) higher than those of Nistari_ silkworm eggs(P<0.05). 【Conclusion】There is certain correlation between ESI and HR of silkworm Nistari varieties. The differe-nces in expression of four genes, BmHE I, BmHE II, Chitinase and Period, are the molecular basis for the differences in HR between silkworm varieties of Nistari- and Nistari+.
        Key words: sailkworm(Bombyx mori); Nistari varieties; egg shape index; hatching rate; hatching related genes
        Foundation item:National Natural Science Foundation of China(31372376,31672490);Jiangsu Natural Science Foundation(BK20141285)
        0 引言
        【研究意義】家蚕(Bombyx mori)是一种重要的鳞翅目经济昆虫,在农业经济体系
中占据重要地位。孵化率是衡量蚕种质量的一项关键性指标,其影响因素包括蚕种品质、制种季节、蚕卵营养、冷藏时间和催青条件等(李燕飞等,2018;高海青等,2019),其中蚕种的卵形及卵重等品质指标对蚕卵孵化率有明显影响(潘明德等,2019)。因此,研究分析家蚕卵形指数对孵化率的影响及其分子机理,对提高蚕种孵化率、蚕种选育与保存具有重要意义。【前人研究进展】昆虫胚胎孵化后才能进行后续的生长发育(Young et al.,2000;Bowles et al.,2008),除了温度、湿度和光照等外部条件对昆虫的胚胎发育有影响外,其内部的基因表达调控网络也会对昆虫孵化及后期发育有显著影响(张艳,2020)。高颂等(2012)研究发现,几丁质酶(Chitinase)主要在昆虫表皮和中肠中表达,与幼虫蜕皮、化蛹和成虫羽化及翅的延展有关。Tao等(2017)研究表明,大多数昆虫的孵化过程受生物钟调控机制严格调节,其中周期蛋白基因(Period)在生物钟信号通路中扮演重要角。家蚕孵化也呈明显的时间节律性,故推测其受Period基因的调控。至今,有关蚕卵孵化的研究已有较多报道。Lu等(2010)在蚕卵孵化过程中发现,消化液从成熟胚中分泌溶解并软化卵壳,从而有利于蚁蚕孵化。Tang等(2012)研究表明,在蚕卵孵化过程中BmHE I和BmHE II两种孵化酶基因的表达趋势与其孵化进程基本一致。Tao等(2017)研究发现,家蚕蚁蚕从含有大量几丁质的卵壳中孵化出来,需依赖几丁质酶将几
丁质降解为寡糖,即蚕卵的孵化过程需要几丁质酶参与。【本研究切入点】已有研究证实禽蛋孵化率与蛋形指数(禽蛋长轴与短轴间的比值)有关。朱振鹏等(2013)研究认为,扬州鹅种蛋的蛋形指数在1.32~1.46时其孵化率最高;张楠楠等(2014)研究发现,肉杂鸡种蛋的蛋形指数在1.25~1.30和1.31~1.37 时孵化效果较好,而在1.19~1.24时孵化效果较差。但针对家蚕卵形指数与其卵孵化率间的相关研究至今未见报道。【拟解决的关键问题】调查家蚕Nistari品种野生型正常形卵(Nistari+)及其新突变体纺锤形卵(Nistari-)的卵形指数、孵化率、受精率和转青死卵率,分析家蚕卵形指数对孵化率的影响规律,以及孵化酶基因(BmHE I和BmHE II)、几丁质酶基因(Chitinase)和周期蛋白基因(Period)在不同卵形间的表达规律,为阐明家蚕卵形指数与孵化率的相关性及其分子机理打下基础。
        1 材料与方法
        1. 1 试验材料
        家蚕Nistari品种的野生型正常形品种Nistari+(图1-A)及其新突变体纺锤形品种Nistari-(图1-B)由江苏科技大学/中国农业科学院蚕业研究所保存提供。其中,Nistari+品
种的蚕卵呈椭圆形,Nistari-品种的蚕卵呈纺锤形。2个品种的蚕卵孵化后,幼虫在25 ℃、相对湿度(65±5)%、12 h光照/12 h黑暗的条件下桑叶饲养,上蔟结茧、化蛹、羽化产卵和蚕卵催青等按常规管理条件进行操作。
        1. 2 品种间卵形指数及孵化率调查
        以正常产卵的框制种Nistari+和Nistari-蚕卵为材料,以蛾圈为单位,每个蛾圈调查50粒卵,调查10个蛾圈。在Leica体视显微镜下测量蚕卵的纵径和横径,计算卵形指数。调查后的蚕种按常规催青条件进行催青,待蚕卵完全孵化后,统计不同品种蛾圈内的总卵数、孵化卵数、受精卵数和转青死卵数,计算蚕卵的孵化率、受精率和转青死卵率。3次重复,取平均值,比较Nistari+和Nistari-品种间的卵形指数和孵化率差异。
        卵形指数=纵径长度(长轴)/横径长度(短轴)
        孵化率(%)=孵化蚕卵数/受精卵数×100
        受精率(%)=受精卵数/总卵数×100
        转青死卵率(%)=(受精卵数-孵化卵数)/受精卵数×100
        1. 3 品种内卵形指数及孵化率调查xujiao
        以正常产卵Nistari+和Nistari-散卵为材料,每个供试品种取3000粒卵,按1.2的方法计算卵形指数后放入预先标记好的不同卵形指数小组内,每组100粒卵。调查后的蚕种按常规催青条件进行催青,待蚕卵完全孵化后,按1.2的方法调查不同卵形指数小组内的总卵数,统计孵化卵数、受精卵数和转青死卵数,计算蚕卵的孵化率、受精率和转青死卵率。3次重复,取平均值,分别分析Nistari+蚕卵及Nistari-蚕卵不同卵形指数与孵化率、受精率和转青死卵率的关系,并进行相关分析。