长期高碱胁迫下凡纳滨对虾基因表达差异研究

Transcriptomic Analysis of Gene Expression of Litopenaeus vannamei during Long-Term Exposure to High Alkaline Water

凡纳滨对虾(Litopenaeus vannamei)具有较强的环境适应能力,对盐碱水环境有一定的耐受性,但在高pH、高碱环境下的存活率不稳定。为探究凡纳滨对虾对长期高碱胁迫的响应机制,本研究以低碱对照组(LSW:碳酸盐碱度为3 mmol/L,盐度为6,pH为8.1)和高碱胁迫组(AW:碳酸盐碱度为10 mmol/L,盐度为6,pH为8.8)养殖42 d的凡纳滨对虾肠道和鳃组织作为实验材料,通过Illumina平台进行转录组测序,对测序数据进行拼接、注释,进而筛选、分析高碱胁迫下的差异表达基因及调控通路并进行定量PCR验证。结果显示,2个组织共同差异表达基因有243个,其中,98个表达上调,145个表达下调。肠道中差异表达基因主要集中在糖代谢、碳水化合物消化吸收、胆汁分泌、ABC跨膜转运、紧密连接以及免疫调节等途径。鳃中差异表达基因主要集中在谷胱甘肽代谢、碳酸氢盐转运、精氨酸合成、糖代谢以及离子转运等相关途径。进一步筛选获得10个最显著的差异表达基因,经qRT-PCR验证发现,凡纳滨对虾鳃中碳酸酐酶(CA1、CA10)、蜕皮激素诱导蛋白(Eip74EF)、β-半乳糖基转移酶(UGT8)基因在高碱胁迫下均表达下调,而Na^(+)/K^(+)-ATPase-α(NKA-α)、Na+/K+transporting ATPase interacting(NKAIN)、氨转运蛋白(Rhbg)、苹果酸脱氢酶(MDH)等基因表达上调,与转录组表达趋势一致,推测其可能参与了对虾高碱胁迫下的应激响应。凡纳滨对虾表现出较强的高碱适应性,可能是通过下调鳃中CA的表达,补偿体内碱中毒,上调Rh氨转运蛋白防止氨在体内积累,上调NKA相关基因维持体内渗透平衡;但蜕皮激素诱导蛋白(Eip74EF)显著下调,推测其蜕皮功能受到影响。本研究为深入探讨凡纳滨对虾在长期高碱胁迫条件下的生理响应机制提供了基础数据。

The total saline-alkaline land area in China is approximately 99.13 million hectares,distributed throughout northern China,coastal areas,and areas along the Huanghe River.About 45.87 million hectares of saline-alkaline water areas are spread around these lands,most of which are athalassic waters characterized by a high pH value above 8.8,associated with high-carbonate alkalinity and various types of ions imbalances.The saline-alkaline land and water cannot be directly used for agriculture,and most of them are arid.The development of aquaculture in saline-alkaline land is not only beneficial to expanding the aquaculture area but also can restore the saline-alkaline soil,which is of great significance to food security and ecological restoration.Saline-alkaline aquaculture is one of the main inland aquaculture models developed in the past ten years.With the maturity of aquaculture technologies,the saline-alkaline aquaculture area has expanded year by year,which has brought earnings to local farmers.China has abundant saline-alkaline water resources.The high pH and high-carbonate alkalinity of these waters restrict the survival,growth,and reproduction of aquatic animals.Litopenaeus vannamei is highly resistant to stress and has a certain tolerance to saline-alkaline water.Under short-term saline-alkaline stress,the expression of the carbonic anhydrase(CA),Na+/K+-ATPase,and other ion-regulated genes of L.vannamei were induced,and the acid-base and osmolality balance were determined by strengthening ion regulation.At present,relatively few studies on gene regulation of L.vannamei under long-term stress have been performed.Although L.vannamei farming has been successful in saline-alkaline water,the survival rate is unstable,and there are few reports on the selective breeding of L.vannamei tolerant to salinity and alkalinity.Through independent innovation,a family-based"multi-trait compound breeding technology for aquatic animals"has been established in China.These techniques have laid a good foundation for developing improved L.vannamei strains.To effectively utilize saline-alkaline water resources,it is urgent to conduct L.vannamei salt-alkali-tolerant breeding and promote the healthy development of the saline-alkaline aquaculture industry.L.vannamei has strong environmental adaptability and relatively high tolerance to saline-alkaline water.It is one of the main species of saline-alkaline aquaculture.However,its survival rate in high pH and high-alkaline environments is not stable.To explore the response mechanism to long-term high-alkaline stress,L.vannamei was exposed to low-alkaline water as the control group(LSW,carbonate alkalinity of 3 mmol/L,salinity of 6,pH of 8.1)and to high-alkaline stress(AW,carbonate alkalinity of 10 mmol/L,salinity of 6,pH of 8.8)for 42 days.The intestine and gill of L.vannamei raised for 42 days were used as the experimental materials.Transcriptome sequencing was performed using the Illumina platform.After splicing analysis and gene annotation,the differentially expressed genes and regulatory pathways regulated under high-alkaline stress were screened and analyzed,with further verification by qRT-PCR.The results showed 243 differentially expressed genes in both tissues,of which 98 were up-regulated and 145 were down-regulated.The differentially expressed genes in the intestine were enriched for glucose metabolism,carbohydrate digestion and absorption,bile secretion,ABC transmembrane transport,and tight junction related pathways.The differentially expressed genes in gills were enriched for glutathione metabolism,bicarbonate transport,arginine synthesis,sugar metabolism,and ion transport related pathways.The ten most significant differentially expressed genes were further studied and verified by qRT-PCR.Carbonic anhydrase(CA1,CA10),ecdysone-inducible protein(Eip74EF),andβ-galactosyltransferase(UGT8)genes in gills were down-regulated.However,the expression of Na^(+)/K^(+)-ATPase-α(NKA-α),Na+/K+transporting ATPase interacting(NKAIN),ammonia transporter(Rhbg),and malate dehydrogenase(MDH)were up-regulated under high-alkaline stress.The transcriptome expression pattern and qRT-PCR results were consistent.We speculated that these genes may be involved in the shrimp stress response to high-alkaline stress.L.vannamei showed a relatively strong high-alkaline tolerance,which may be compensated by down-regulating the expression of CA to prevent alkalosis,up-regulating Rhbg to prevent ammonia accumulation and NKA-related genes to maintain the osmotic balance.The ecdysone function was probably affected as the Eip74EF gene was down-regulated.This study provides basic data for further analyzing the physiological response mechanisms of L.vannamei under long-term highly alkaline stress.