Expression Pattern of Silkworm 30K Protein BmLP1 and Its Role in Embryonic Development

ZHANG Yan; DONG Zhaoming; XIA Jianhua; LINGHU Qingqing; WAN Na; ZHANG Renyi; LU Zhongyan; LIN Ying; ZHAO Ping

doi:10.13718/j.cnki.xdzk.2024.10.008

2024 Volume 46 Issue 10

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ZHANG Yan, DONG Zhaoming, XIA Jianhua, et al. Expression Pattern of Silkworm 30K Protein BmLP1 and Its Role in Embryonic Development[J]. Journal of Southwest University Natural Science Edition, 2024, 46(10): 99-106. doi: 10.13718/j.cnki.xdzk.2024.10.008

Citation:

ZHANG Yan, DONG Zhaoming, XIA Jianhua, et al. Expression Pattern of Silkworm 30K Protein BmLP1 and Its Role in Embryonic Development[J]. Journal of Southwest University Natural Science Edition, 2024, 46(10): 99-106. doi: 10.13718/j.cnki.xdzk.2024.10.008

Expression Pattern of Silkworm 30K Protein BmLP1 and Its Role in Embryonic Development

Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Southwest University/Biological Science Research Center, Academy for Advanced Interdisciplinary, Chongqing 400715, China

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Received Date: 05/09/2023
Available Online: 20/10/2024
MSC: S881.2

Abstract

30K proteins are abundant in silkworm hemolymph and egg. They are involved in nutrition, immunity, inhibition of apoptosis and other functions. We studied the expression of 30K proteins to better understand their functions. In this research, bioinformatic analysis showed that all the Bmlp1, Bmlp2, Bmlp3 and Bmlp7 genes have signal peptides. The sequence similarity between Bmlp1 and other 30K protein genes is less than 50%. The result of semi-quantitative RT-PCR showed that Bmlp1 was highly expressed from day 3 of fifth instar larvae to day 1 of pupae, and then decreased. By Western blotting assay, we found that the BmLP1 antibody could only bind with BmLP1 protein, not BmLP2, BmLP3 and BmLP7 proteins, suggesting that the antibody has good specificity. The Western blotting result showed that BmLP1 was abundant from day 5 of fifth instar larvae to day 4 of pupae, and then decreased, whereas BmLP1 was firstly observed after day 2 of wandering, then increased gradually, and reached a maximum level in the moth. This is because that ovary tubes grew quickly from day 2 of wandering, and BmLP1 and other nutrients-storage proteins were transported into the ovary tubes and accumulated in the unfertilized eggs. Immmunohistochemistry result showed that BmLP1 was located in the yolk granules on day 6 of egg and in the gut before the hatching. We incubated BmLP1 with the new hatched larvae extract in vitro, and found that the extract could degraded the majority of BmLP1, indicating that there may be one protease in the gut responsible for the degradation of BmLP1. BmLP1 could provide the energy and amino acid resources for the hatching of baby silkworm larvae. This study explored the changing pattern of silkworm 30K protein BmLP1 in the fat body, hemolymph, ovary and egg at different developmental stage, laying the foundation for understanding the function of BmLP1 in the silkworm embryonic development.
- silkworm (Bombyx mori),
- 30K protein,
- BmLP1,
- embryonic development,
- hemolymph

References

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家蚕是我国的重要经济昆虫，已经有5 000年的驯化历史. 近年来，家蚕成为了典型的鳞翅目模式昆虫，对家蚕的基础研究可为研究其他鳞翅目昆虫提供重要的理论支撑. 家蚕具有开放式血淋巴循环系统，组织器官浸润于血淋巴中. 血淋巴在营养物质转运、废物运输等方面发挥着至关重要的作用. 家蚕血淋巴中有几百种蛋白质，这些蛋白质主要分为2大类：营养储存蛋白和免疫相关蛋白^[1]. 营养储存蛋白主要包括载脂蛋白、贮藏蛋白、卵黄原蛋白和30K蛋白. 30K蛋白是序列高度同源且分子量在30K左右的一群蛋白，在家蚕血液和胚胎中含量非常高^[2-6]. 家蚕30K蛋白的功能研究将有助于我们理解30K蛋白在血液和胚胎中的作用.

2007年，SUN等^[7]利用家蚕的EST数据对30K蛋白基因进行了鉴定，共鉴定到10个基因. 2012年，ZHANG等^[8]利用家蚕基因组精细图序列数据，确定家蚕中共有46个基因编码30K蛋白. 根据进化分析和结构域分析，可以将30K蛋白分为3个家族：ENF肽结合蛋白、富含丝氨酸和苏氨酸的30K蛋白和典型30K蛋白. 典型30K蛋白分子量约为30 kDa，共有24个成员. 对4龄和5龄家蚕血液进行蛋白质组学分析，结果表明典型30K蛋白在5龄家蚕血液中高量存在，尤其是BmLP1和BmLP2^[1]. 已有研究表明，BmLP1有多种生物学功能，在胚胎的正常发育中具有重要作用^[9]. BmLP1可以与糖结合，如甘露糖、葡萄糖、N-乙酰葡糖胺^[10]和葡聚糖，葡聚糖是病原体表面携带的重要糖分子，BmLP1可以结合葡聚糖，暗示其可能参与了昆虫的免疫^[11-12]；YE等^[13]证实BmLP1可以与血细胞膜结合，通过促进血细胞包囊及黑化反应参与家蚕免疫；BmLP1可以结合EcR-B1，从而抑制蜕皮激素诱导的细胞凋亡^[14]. ZHANG等^[9]对BmLP7进行了详细研究，发现BmLP7主要由5龄家蚕的脂肪体合成，然后分泌进入血液中，再转运进入卵巢中，最终在胚胎发育过程中被胚胎肠腔内的酶降解，为胚胎发育提供能量和氨基酸. 近期有研究表明，BmVgR(家蚕卵黄原蛋白受体)可以与BmLP1结合，将BmLP1转运进入卵巢. 2016年，WANG等^[15]从5龄家蚕消化液中纯化获得碱性丝氨酸蛋白酶P-IIc，该酶在胚胎后期表达，将该蛋白酶与不同30K蛋白孵育后，发现其具有强烈水解30K蛋白的功能.

30K蛋白成员众多，且在生物学功能上呈现出丰富的多样性. 前期研究了30K蛋白BmLP7的表达模式与功能，为了系统地比较与分析不同30K蛋白的功能差异，有必要对BmLP1等其他30K蛋白进行研究. 本研究利用半定量RT-PCR，Western blotting，免疫组化等技术，对家蚕30K蛋白BmLP1在不同组织器官中的表达模式进行调查和分析，旨在揭示家蚕BmLP1蛋白的合成、转运与利用情况，阐明家蚕30K蛋白在胚胎发育中的功能.

1. 材料与方法

1.1. 材料与试剂

家蚕大造为本研究的实验材料，由西南大学提供. 大造采用常规饲喂方法饲养，饲喂的相对温度为25 ℃，相对湿度为60%.

Trizol试剂购于Invitrogen公司；dNTPs，Oligo (dT)，M-MLV反转录酶购于Promega公司；羊抗兔二抗购于Sigma公司；Tubulin抗体、FITC染色液和抗荧光淬灭封闭液购于碧云天；DAB显色试剂盒购于北京中杉金桥；PVDF膜购于Roche；苯基硫脲、考马斯亮蓝、甘氨酸等试剂购于上海生工；松油醇、二甲苯、磷酸二氢钠、磷酸氢二钠、甲醇及冰醋酸等试剂购于重庆化学试剂厂.

1.2. 氨基酸多序列比对分析

在Silkdb数据库(http://silkworm.swu.edu.cn/silkdb/)下载30K蛋白BmLP1，BmLP2，BmLP3和BmLP7的氨基酸序列^[16]，利用ClustalX 1.83软件进行多序列比对分析^[17]，并利用GeneDoc软件进行手动校正.

1.3. 30K蛋白基因的RT-PCR检测

收集不同发育时期的家蚕脂肪体材料放于-80 ℃冰箱中保存备用. 家蚕的发育阶段共8个，分别为：5龄第3 d，5龄第5 d，5龄第7 d，上蔟第2 d，化蛹第1 d，化蛹第4 d，化蛹第7 d和化蛾第1 d. 利用Trizol试剂提取脂肪体的总RNA，反转录为cDNA. 利用cDNA为模板进行PCR扩增，扩增所用引物见表 1，扩增条件为：94 ℃预变性4 min；94 ℃变性40 s；54 ℃退火40 s；72 ℃延伸40 s；循环28次；72 ℃再延伸10 min；最后置于12 ℃保存.

1.4. 30K蛋白的SDS-PAGE及Western blotting分析

家蚕发育至5龄第1 d时，鉴定性别，取雌性家蚕作为实验材料. 在5龄期和上蔟期，先用解剖针扎家蚕腹足，收集血液，并加入少量的苯基硫脲防止血液黑化，再取家蚕的卵巢组织存放于-80 ℃冰箱中. 在蛹期和蛾期，将家蚕固定于蜡盘上，用20 μL枪头吸取血液置于离心管中保存，再取其卵巢组织保存于-80 ℃冰箱中. 血液样品用0.1 mol/L PBS溶液稀释5倍待用. 卵巢样品采用液氮研磨的方法研磨成粉末状，加入适量的0.1 mol/L PBS溶液溶解.

利用Brandford法对血液样品和卵巢样品进行定量分析，并以牛血清白蛋白(BSA)绘制标准曲线. 根据定量结果，取2 μg蛋白质样品进行聚丙烯酰胺凝胶电泳(SDS-PAGE)分析，凝胶浓度为12.5%. 将血液和卵巢样品进行聚丙烯酰胺凝胶电泳后，通过湿转法将蛋白转移至PVDF膜上，转膜条件为200 V，转膜时间为30 min. 待转膜完成后，将膜放置在培养皿中，用5%的脱脂奶粉封闭1 h后，倒掉封闭液，加入一抗溶液孵育2 h，用TBST溶液清洗4次，再加入二抗溶液孵育2 h，再用TBST溶液清洗4次. 最后将PVDF膜浸泡在显色液中，避光反应5 min，放入曝光仪中曝光.

1.5. 免疫组织化学定位分析

当蚕卵发育至第6 d和第10 d时，取适量的卵放入10% KOH溶液中软化，然后置于Smith固定液中24 h，接着用乙醇梯度脱水，用松油醇处理，使之变得透明. 最后，将处理好的蚕卵包埋进石蜡中，用石蜡切片机进行切片，切片厚度设置为5 μm. 石蜡切片经过脱蜡、乙醇复水、去除内源性过氧化物酶、抗原修复、封闭、一抗孵育和二抗孵育等处理后，用DAPI和DAB显色液进行显色，在显微镜下观察并拍照.

1.6. 蚁蚕粗提物对BmLP1的降解

蚁蚕粗提物对BmLP1降解实验的反应体系如下：BmLP1的对照组中只加入了BmLP1蛋白，最终体积用缓冲液补足；蚁蚕粗提物组中只加入了蚁蚕粗提物，最终体积用缓冲液补足；BmLP1与蚁蚕粗提物孵育的实验组中加入了蚁蚕粗提物与BmLP1，其质量比为3∶1. 缓冲液为100 mmol/L的Tris-HCl，pH值为8.8. 对照组与实验组都在37 ℃下孵育2 h，保证蚁蚕粗提物中的酶与BmLP1充分反应. 反应结束后，取相同体积的量进行SDS-PAGE电泳检测降解效果.

3. 讨论与结论

3.1. 讨论

在家蚕的末龄幼虫中，血液重量一般占体质量的1/4^[18]. 在5龄期幼虫的血液中，30K蛋白是含量最大的一群蛋白质之一^{[4-5, 9]}. 在其他鳞翅目昆虫，如烟草天蛾、蓖麻蚕、琥珀蚕和印度柞蚕中也鉴定到了30K蛋白的同源基因^[8]，但这些物种的血液中30K蛋白的表达量并不高.

本研究选取了在家蚕5龄第7 d血液中含量最高的典型30K蛋白BmLP1进行研究，发现Bmlp1基因在脂肪体中高量表达，表明BmLP1和其他典型30K蛋白均在脂肪体中合成^[19]. 由于Bmlp1编码的蛋白质具有信号肽，是一种可分泌蛋白，在脂肪体中表达的Bmlp1基因分泌进入血液中，因此，在5龄期和蛹前期，血液中BmLP1蛋白含量都较高. 但在蛹后期和蛾期，BmLP1蛋白逐渐减少，与此同时，卵巢中的BmLP1却开始增加. 卵巢从蛹期开始急剧生长，卵巢管冲破卵巢膜，游离于体腔中. 卵在卵巢管中形成，并从血液中摄取营养以支持其在蛹期的快速生长. BmLP1被卵巢从血液中吸收并积累到卵中，作为重要的营养物质. 这些转运进卵巢的BmLP1在胚胎发育过程中一直不被利用，直到胚胎后期才开始减少. XU等^[21]的研究表明BmLP1是通过BmVgR转运进卵巢的. 免疫组织化学定位分析结果表明，BmLP1蛋白一直储存在卵黄颗粒中，当胚子发育成熟后，胚子将含有BmLP1的卵黄颗粒吞入肠道内. 蚁蚕粗提物孵育实验结果表明，蚁蚕肠道内存在可以降解BmLP1的蛋白酶，最终将BmLP1降解为氨基酸. 这些结果与之前报道的BmLP3和BmLP7的结果相似^[8]，暗示了典型30K蛋白的共同特性：合成于脂肪体，分泌入血液，转运进卵巢，贮存在卵中，在蚁蚕孵化前被降解掉.

孵育实验结果表明，蚁蚕粗提物可以将大部分BmLP1水解. 2016年，WANG等^[15]从家蚕的5龄幼虫肠液中纯化到一种碱性丝氨酸蛋白酶P-IIc，该蛋白酶最适pH值为11，最适温度为25 ℃. 碱性丝氨酸蛋白酶P-IIc是一个广谱性蛋白酶，可以水解大部分桑叶蛋白质. 另外，该蛋白酶在胚胎后期具有很高的表达量，可以水解卵黄磷蛋白、ESP蛋白和30K蛋白. 值得注意的是，该蛋白酶可以水解大部分BmLP1蛋白，只能部分水解BmLP2、BmLP3、BmLP7和BmLP8^[15]，表明其对不同底物的活性不同. 因此，认为家蚕胚胎后期，肠中的丝氨酸蛋白酶P-IIc是导致30K蛋白BmLP1降解的主要水解酶.

3.2. 结论

本研究发现30K蛋白Bmlp1基因在5龄幼虫和蛹期的脂肪体中高量表达，分泌进入血液中. 随着蛹期卵巢管的萌发，BmLP1逐渐从血液转运进卵巢. BmLP1蛋白一直储存在卵巢管的卵中，最终定位于卵黄颗粒中储存. 当胚子发育成熟后，胚子将含有BmLP1的卵黄颗粒吞入肠道内. 肠道内存在可以降解BmLP1的蛋白酶，最终将BmLP1降解为氨基酸，为胚胎的发育提供营养.

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Expression Pattern of Silkworm 30K Protein BmLP1 and Its Role in Embryonic Development