摘要
球孢白僵菌(Beauveria bassiana)作为一种常见的致昆虫疾病的真菌,广泛应用于农林害虫和城市昆虫的生物防治。几丁质酶Bbchit1基因为球孢白僵菌侵染过程中分解昆虫体壁的一个重要基因。为分析球孢白僵菌XJBB-01株重组几丁质酶(r Bbchit1)的杀蜱活性,本实验利用PCR扩增球孢白僵菌XJBB-01株Bbchit1基因的编码区序列,经原核系统表达重组Bbchit1蛋白(r Bbchit1),采用镍亲和层析法纯化rBbchit1,并经SDS-PAGE检测rBbchit1的表达与纯化效果。利用软件分析Bbchit1基因及其编码蛋白Bbchit1的生物信息学特征。采用分光光度法测定纯化蛋白的浓度。结果显示,Bbchit1基因长1047 bp,编码348个氨基酸。rBbchit1是一种酸性、稳定的亲水性分泌蛋白,有信号肽,无跨膜域,二、三级结构均以无规则卷曲为主,亲水性低,抗原性较弱。SDS-PAGE结果显示,rBbchit1主要在包涵体中出现39 ku的目的条带,纯化后条带单一,纯化的rBbchit1浓度为0.7 mg/mL。将拉合尔钝缘蜱分别浸入含1mg/mL和3mg/mL的rBbchit1中25℃处理24 h后,每天定时利用体视显微镜观察并记录各组蜱的形态变化和死亡数量,持续观察10 d后经扫描电镜观察死亡蜱的变化。体视显微镜观察结果显示,rBbchit1作用拉合尔钝缘蜱后1 d,该蜱活动减少、体壁被降解、出血,作用后3 d死亡的拉合尔钝缘蜱全身出现大量出血点,作用后5 d正在蜕皮的拉合尔钝缘蜱还未完全蜕皮即死亡脆化,外皮褶皱密布出血点,腹部有白色晶体析出。不同浓度处理的拉合尔钝缘蜱形态学特征无明显区别;1 mg/mL与3 mg/mL的rBbchit1对拉合尔钝缘蜱的最高杀灭率分别为50%与68.75%;扫描电镜观察显示,不同浓度的rBbchit1处理后,拉合尔钝缘蜱的体壁由原本的光滑完整变为松散粗糙不平的塌陷状。无rBbchit1作用的对照组蜱均键活。表明rBbchit1可以通过破坏拉合尔钝缘蜱的体壁结构致其死亡,并使其蜕皮困难。本研究首次探究rBbchit1在实验室条件下体外侵染拉合尔钝缘蜱的生理过程,为真菌-蜱虫互作机制及孢白僵菌对拉合尔钝缘蜱侵染机制的研究提供基础数据。
Beauveria bassiana,a prevalent entomopathogenic fungus,is extensively utilized in the biological control of agricultural,forestry,and urban insect pests.The chitinase gene Bbchit1 plays a crucial role in the degradation of insect cuticles during Beauveria bassiana infection.To evaluate the insecticidal activity of recombinant chitinase(rBbchit1)from Beauveria bassiana strain XJBB-01,the coding sequence of Bbchit1 was amplified by polymerase chain reaction and subsequently expressed in a prokaryotic system.The recombinant protein was purified via nickel-affinity chromatography,and its expression and purification efficiency were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Bioinformatics analysis was performed to characterize the Bbchit1 gene and its encoded protein.The concentration of the purified protein was determined by spectrophotometrically.The results indicated that Bbchit1 is 1047bp in length,encoding a 348-amino acid protein.Bbchit1 is an acidic,stable,hydrophilic secretory protein with a signal peptide and without transmembrane domain.Its secondary and tertiary structures are predominantly composed of random coils,exhibiting low hydrophilicity and weak antigenicity.SDS-PAGE revealed a 39ku target band primarily present in inclusion bodies,with a single purified band and a final rBbchit1 concentration of 0.7mg/mL.Ornithodoros lahorensis were immersed in rBbchit1 solutions with concentrations of 1mg/mL and 3mg/mL,respectively,at 25℃for 24 hours,followed by daily stereomicroscopic observation of morphological changes and mortality over 10 days.Scanning electron microscopy(SEM)was used to examine structural alterations in deceased ticks.Stereomicroscopic analysis showed that,within 1 day of rBbchit1 exposure,the ticks exhibited reduced mobility,cuticle degradation,and hemorrhage.By day 3,extensive hemorrhagic spots were observed on deceased ticks.On day 5,molting ticks died before completing ecdysis,displaying brittle cuticles,dense hemorrhagic folds,and white crystalline deposits on the abdomen.No significant morphological differences were observed between different rBbchit1 concentrations.The highest mortality rates were 50%at 1mg/mL and 68.75%at 3mg/mL.SEM revealed that rBbchit1 treatment transformed the originally smooth and intact tick cuticle into a collapsed,loose,and rough structure.Control ticks without rBbchit1 exposure remained viable.These findings indicate that rBbchit1 induces mortality in Ornithodoros lahorensis by disrupting cuticle integrity and impeding molting.This study is the first to investigate the physiological effects of rBbchit1 on Ornithodoros lahorensis under laboratory conditions,providing foundational data and novel insights into the fungal-tick interaction mechanism and the infection dynamics of Beauveria bassiana against Ornithodoros lahorensis.
作者
姜冰冰
刘丹丹
朱慧茹
马思楠
李龙涛
黄景仁杰
王永青
张泽田
李欣如
黄洁
周雨芊
崔舒溶
陈奇
张佳祎
艾日登才次克
巴音查汗·盖力克
张伟
JIANG Bing-bing;LIU Dan-dan;ZHU Hui-ru;MA Si-nan;LI Long-tao;HUANG Jing-renjie;WANG Yong-qing;ZHANG Ze-tian;LI Xin-ru;HUANG Jie;ZHOU Yu-qian;CUI Shu-rong;CHEN Qi;ZHANG Jia-yi;Airidengcaicike;GAILIKE Bayinchahan;ZHANG Wei(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Xinjiang Key Laboratory of New Drug Research and Development for Herbivores,Urumqi 830052,China;The Xinjiang Uygur Autonomous Region Animal Health Inspection Institute,Urumqi 830011,China)
出处
《中国预防兽医学报》
北大核心
2025年第6期577-584,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
新疆维吾尔自治区自然科学基金面上项目(2022D01A166)
国家自然科学基金项目(32060803)
新疆维吾尔自治区天山英才创新团队-生物媒介及其传播人畜共患虫媒病研究创新团队(2023TSYCTD0008)
新疆维吾尔自治区兽医学特色学科和“新疆草食动物新药研究与创制”重点实验室开放课题(XJCDVM-HDRC-S202412)。
关键词
球孢白僵菌
几丁质酶
拉合尔钝缘蜱
杀蜱活性
Beauveria bassiana
chitinase
Ornithodoros lahorensis
tick-killing activity
