家蚕己糖激酶基因进化分析及其多转录本在胚胎早期发育过程中的表达特性

Phylogenetics of hexokinase gene and expression characteristics of its multiple transcripts in the early embryonic stage of Bombyx mori

【目的】明确家蚕(Bombyx mori)二化性品系胚胎早期发育过程中己糖激酶基因(BmHK)不同转录本的表达特性,为深入探究BmHK基因各转录本的生物学功能及揭示其在家蚕胚胎早期发育中的作用机制提供理论依据。【方法】从NCBI检索并下载家蚕等12种昆虫的HK氨基酸序列,采用MEGA 7.0的邻接法(NJ)构建基于HK氨基酸序列相似性的系统发育进化树。以家蚕二化性品系秋丰活化越年蚕卵(丙2胚胎)为材料,分别制备非滞育命运卵(ND)、滞育命运卵(DD)和即时浸酸卵(IA),采用实时荧光定量PCR检测BmHK基因各转录本在家蚕二化性品系胚胎早期发育过程中的表达特性。【结果】BmHK基因属于HSP70-actin家族,存在4个不同的转录本,其长度分别为4610、4608、2818和1520 bp,依次命名为BmHK-a、BmHK-b、BmHK-c和BmHK-d。BmHK-a在3种蚕卵胚胎早期发育过程中整体上呈先升高后降低的变化趋势,且各时间点均表现为IA蚕卵>ND蚕卵>DD蚕卵,IA蚕卵中的BmHK-a相对表达量于发育2 h达峰值,在ND蚕卵和DD蚕卵中于发育48 h达峰值,此后其表达快速下调,至发育96 h均处于较低水平;BmHK-b在3种蚕卵中的表达水平整体上表现为IA蚕卵>ND蚕卵>DD蚕卵,3种蚕卵中的BmHK-b相对表达量变化趋势基本一致,均随发育时间推移呈逐渐上升趋势;BmHK-c在3种蚕卵中的表达差异明显,初产蚕卵中已有较高的表达水平,受精后迅速升高,至发育72 h后BmHK-c在3种蚕卵中的相对表达量再次升高;BmHK-d在3种蚕卵中的相对表达量整体上呈先升高后降低的变化趋势,且各时间点均表现为ND蚕卵>IA蚕卵>DD蚕卵,但IA蚕卵中BmHK-d表达峰值出现的时间早于DD蚕卵和ND蚕卵。【结论】BmHK-a和BmHK-d主要在家蚕胚胎早期发育(产卵或浸酸后3 d)过程中发挥重要作用;BmHK-b虽然也参与家蚕胚胎早期发育过程,但不是主要的糖代谢相关酶;BmHK-c与家蚕胚胎早期发育关系密切,通过糖酵解为胚胎发育提供能量。

【Objective】To clarify the expression characteristics of the different transcripts of the hexokinase gene(BmHK)in the early embryonic development stage of bivoltine silkworm(Bombyx mori),and in order to provide a theoretical basis for further exploring the biological functions of each transcript of the BmHK gene and revealing the mechanism of ac-tion in the early embryonic development of silkworm.【Method】The HK amino acid sequences of 12 species of insects in-cluding B.mori were obtained and downloaded from NCBI,and a phylogenetic tree based on the similarity of HK amino acid sequences was constructed using the neighbor-joining method(NJ)of MEGA 7.0.The non-diapause-destined eggs(ND),diapause-destined eggs(DD)and immediately acid-treated eggs(IA)were prepared using Qiufeng activated hiber-nating eggs(C-2 embryos)of the silkworm bivoltine strain.Real-time fluorescence quantitative PCR was used to detect the expression characteristics of each transcript during the early embryonic development of silkworm.【Result】The BmHK gene belonged to the HSP70-actin family,and there were 4 different transcripts with lengths of 4610,4608,2818,and 1520 bp,respectively named BmHK-a,BmHK-b,BmHK-c and BmHK-d.BmHK-a showed an overall trend of increasing first and then decreased during the early embryonic development of the three kinds of silkworm eggs.At each time point,it was expressed as IA silkworm eggs>ND silkworm eggs>DD silkworm eggs,in IA silkworm eggs the relative expres-sion of BmHK-a reached a peak at 2 h of development,and reached a peak at 48 h of development in ND silkworm eggs and DD silkworm eggs.After that,its expression was rapidly down-regulated and remained at a low level until 96 h of de-velopment;expression level of BmHK-b in silkworm eggs in three species was as follows:IA silkworm eggs>ND silk-worm eggs>DD silkworm eggs.The relative expression levels of BmHK-b in the three silkworm eggs had basically the same trend,and they all showed a gradual upward trend with the development time.The expression of BmHK-c in the three silkworm eggs was greatly different.The expression level in the first-laying silkworm eggs was already high,and it increased rapidly after fertilization.The relative expression level of BmHK-c in the three silkworm eggs at 72 h after deve lopment increased again;the relative expression of BmHK-d in the three silkworm eggs showed an overall trend of first in-creasing and then decreasing,and at each time point it was ND silkworm eggs>IA silkworm eggs>DD silkworm eggs,but the peak of BmHK-d expression in IA silkworm eggs appeared earlier than DD silkworm eggs and ND silkworm eggs.【Conclusion】BmHK-a and BmHK-d mainly play an important role in the early embryonic development of the silkworm(3 d after laying eggs or soaking in acid);although BmHK-b is also involved in the early embryonic development of the silkworm,it is not the main sugar metabolism related enzymes;BmHK-c is closely related to the early embryonic deve lopment of the silkworm,and provides energy for embryonic development through glycolysis.