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1. Identification and characterization of a TLR13 gene homologue from Laodelphax striatellus involved in the immune response induced by rice stripe virus

ZHOU Xue, HU Jia, FU Mei-li, JIN Ping, ZHANG Yun-ye, XIANG Ying, LI Yao, MA Fei

Journal of Integrative Agriculture 2020, 19 (1): 183-192. DOI: 10.1016/S2095-3119(19)62795-4

摘要（200）

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Toll-like receptors (TLRs) are the critical superfamily homologues that initiate sensing of the invasion of pathogens by the Toll pathway. As one of several intracellular nucleic acid-sensing TLRs, TLR13 is activated by an unmethylated motif present in the large ribosomal subunit of bacterial RNA. However, little attention has been paid to the function of TLR13 gene homologue from Laodelphax striatellus (designated as LsToll-13) in the immune response to rice stripe virus (RSV). Herein, LsToll-13 was cloned and characterized using RACE-PCR. Phylogenetic analysis showed that LsToll-13 was clustered with the TLR13 from six insects. Real-time PCR analysis demonstrated that the expression level of LsToll-13 was significantly reduced in L.?striatellus with RSV infection compared with that in the naive strain. When the expression of LsToll-13 was significantly up-regulated at 6 h after bacterial infection, the expression of ribonucleoprotein (RNP) indicated that the RSV titer in the host insect was significantly suppressed. Upon knockdown of LsToll-13, using RNA interference (RNAi) in L.?striatellus, the expression level of RNP was significantly increased with enhanced RSV accumulation, suggesting that LsToll-13 potentially protects L.?striatellus from RSV infection. Taken together, our results indicated that LsToll-13 might be involved in the immune response of L.?striatellus to RSV infection, and provided a new insight into further elucidating the molecular mechanisms of complex pathogen-host interactions and integrative pest management.

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2. Functional identification of C-type lectin in the diamondback moth, Plutella xylostella (L.) innate immunity

LI Jin-yang, LIN Jun-han, G. Mandela FERNáNDEZ-GRANDON, ZHANG Jia-yu, YOU Min-sheng, XIA Xiao-feng

Journal of Integrative Agriculture 2021, 20 (12): 3240-3255. DOI: 10.1016/S2095-3119(21)63650-X

摘要（175）

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C-型凝集素(C-type lectins，CTLs)是一种依赖Ca²⁺的碳水化合物识别蛋白超家族，也是昆虫天然免疫中一类重要的模式识别受体(pattern recognition receptor，PRR)，可介导昆虫的体液免疫和细胞免疫。我们从小菜蛾基因组中克隆到了一个新的含有双碳水化合物识别结构域(carbohydrate-recognition domain，CRD)的C-型凝集素，命名为PxIML。PxIML的开放阅读框长969 bp，编码322个氨基酸残基。该蛋白序列含有一个信号肽和一个带有EPN(Glu₁₂₄-Pro₁₂₅-Asn₁₂₆)和QPD(Gln₂₇₄-Pro₂₇₅-Asp₂₇₆)基序的双碳水化合物识别结构域。表达谱分析发现，PxIML在脂肪体中的表达显著高于血淋巴和中肠。苏云金芽胞杆菌(Bacillus thuringiensis-8010，Bt8010)感染小菜蛾18 h后，PxIML在全虫和脂肪体中的表达受到抑制，而被粘质沙雷氏菌IAE6(Serratia marcescens-IAE6)和毕赤酵母(Pichia pastoris)感染后，PxIML在全虫和脂肪体中的表达显著上升。重组PxIML(rPxIML)能与所测试的病原体相关分子模式(pathogen-associated molecular patterns，PAMPs)，以及肠杆菌IAE5(Enterobacter sp. IAE5)、粘质沙雷氏菌IAE6、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌BL21(Escherichia coli BL21)和Bt8010以依赖Ca²⁺的方式结合，但与毕赤酵母不结合。在Ca²⁺存在的条件下，rPxIML对Bt8010、肠杆菌IAE5和金黄色葡萄球具有较强的凝集活性，但对大肠杆菌BL21的凝集活性弱，对粘质沙雷氏菌IAE6和毕赤酵母不凝集。此外，rPxIML还能与血细胞结合，促进血细胞对包被rPxIML凝胶珠的吸附，增强酚氧化酶活性，促进黑化反应。我们的研究结果表明，PxIML在病原体识别和介导小菜蛾随后的体液免疫和细胞免疫中起着重要作用。

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3. Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae)

SHU Ben-shui, YU Hai-kuo, DAI Jing-hua, XIE Zi-ge, QIAN Wan-qiang, LIN Jin-tian

Journal of Integrative Agriculture 2021, 20 (9): 2471-2482. DOI: 10.1016/S2095-3119(20)63298-1

摘要（163）

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本文选取Actin，EF1α，EF2，GAPDH，RPL3，RPL13，α-TUB和β-1-TUB基因作为候选内参基因；采用实时荧光定量PCR分析了8个候选内参基因在草地贪夜蛾不同龄期，6龄幼虫不同组织，幼虫在不同温度和饥饿处理等实验条件下的转录水平表达量；采用ΔCt，BestKeeper，geNorm，NormFinder和RefFinder对各个候选内参基因进行表达稳定性评估。GeNorm分析结果表明在本研究中不同实验条件下用于分析靶标基因转录水平表达量的最适内参基因个数均为2个。综合分析结果表明草地贪夜蛾不同发育阶段最稳定的内参基因为EF2和RPL13, 6龄幼虫不同组织中最稳定的内参基因为RPL13和β-1-TUB，不同温度处理下三龄幼虫中最稳定的内参基因为EF2和EF1α，饥饿处理条件下三龄幼虫中最稳定的内参基因为RPL3和EF1α，所有样本中较为稳定的内参基因为RPL13和EF1α。本研究为草地贪夜蛾不同实验条件下内参基因选择提供了参考，同时也有助于保证后续靶标基因转录水平表达研究的准确性。

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4. Functional analysis of the orphan genes Tssor-3 and Tssor-4 in male Plutella xylostella

LI Tian-pu, ZHANG Li-wen, LI Ya-qing, YOU Min-sheng, ZHAO Qian

Journal of Integrative Agriculture 2021, 20 (7): 1880-1888. DOI: 10.1016/S2095-3119(21)63655-9

摘要（137）

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孤儿基因是指在其他物种中没有同源序列的一类基因。在此，我们在小菜蛾中鉴定了两个孤儿基因，命名为Tssor-3和Tssor-4。这两个基因都含有一个信号肽序列，表明它们具有分泌蛋白的功能。基于实时荧光定量PCR（qPCR）的表达模式分析表明，这两个孤儿基因均在除睾丸外的雄性生殖腺中特异表达；其表达量在雄成虫时期达到顶峰。免疫荧光实验表明，这两种蛋白均为精液蛋白，暗示它们在调节雄性生殖方面具有潜在的作用。为了进一步探索它们的功能，我们通过RNA干扰（RNAi）下调了这两个基因的表达量，结果表明，干扰后24 h，Tssor-3和Tssor-4的表达量均显著低于对照组。生物测定实验表明，当Tssor-3和Tssor-4基因表达量降低时，小菜蛾的产卵量和子代卵的孵化率均显著下降，表明这两个孤儿基因在小菜蛾雄性育性中起作用。我们的结果为孤儿基因参与雄性生殖调节提供了证据，这对雄性在进化过程中的适应性很重要。

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5. Identification and tissue distribution of odorant binding protein genes in Harmonia axyridis (Coleoptera: Coccinellidae)

QU Cheng, WANG Ran, CHE Wu-nan, LI Feng-qi, ZHAO Hai-peng, WEI Yi-yun, LUO Chen, XUE Ming

Journal of Integrative Agriculture 2021, 20 (8): 2204-2213. DOI: 10.1016/S2095-3119(20)63297-X

摘要（135）

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昆虫嗅觉系统在其寻找寄主、交配和产卵等行为中发挥重要作用。气味结合蛋白（OBPs）参与化学信息素的识别，OBPs识别结合气味分子并将其运送至嗅觉神经元上的气味受体。异色瓢虫（鞘翅目：瓢甲科）作为重要的生防天敌被广泛用于防治多种农林业害虫。本研究中，从异色瓢虫成虫触角和整虫转录组中共鉴定到19个OBP基因，所有OBP基因均具有全长开放阅读框，其编码蛋白中11个属于‘Classic’OBPs，7 个属于‘Minus-C’OBPs ，1 个属于‘Plus-C’OBP。它们编码125-241个氨基酸，相对分子量为13.74KDa-27.75 KDa，等电点范围为4.15-8.80。利用系统发育分析研究了异色瓢虫气味结合蛋白与其他鞘翅目昆虫气味结合蛋白的关系。荧光定量PCR结果表明HaxyOBP2，3，5，8，10，12，13，14和15在雌雄成虫触角中高表达；其中HaxyOBP2，3，5，12和15在触角中的表达量显著高于其他组织，HaxyOBP13和HaxyOBP14在触角和头部的表达量相当，而其他基因在胸、腹、足和翅等非嗅觉组织中高表达。本研究结果为进一步探究异色瓢虫嗅觉系统提供了有价值信息，并将增强异色瓢虫作为生防天敌的应用效果。

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6. Pancreatic triglyceride lipase is involved in the virulence of the brown planthopper to rice plants

YUAN Long-yu, HAO Yuan-hao, CHEN Qiao-kui, PANG Rui, ZHANG Wen-qing

Journal of Integrative Agriculture 2020, 19 (11): 2758-2766. DOI: 10.1016/S2095-3119(20)63188-4

摘要（116）

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The brown planthopper (BPH), Nilaparvata lugens, an important rice insect pest, can enhance its virulence to BPH-resistant rice within as short a span as several generations. Here, we cloned a pancreatic triglyceride lipase (PTL) gene (NlPTL) in N. lugens, and found that its mRNA level was higher in the high virulence population (fed on variety Rathu Heenati, P-RH) than in the low virulence population (fed on variety Taichung Native 1, P-TN1). Knocking down NlPTL caused BPH individuals to spend more time in non-penetration and the pathway phases and less time feeding on the phloem of rice plants; these changes consequently decreased food intake, lipid content, survival rate, and fecundity in the insects. These findings reveal for the first time that PTL in BPH is involved in its virulence to rice plants.

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7. Cuticular protein gene LmACP8 is involved in wing morphogenesis in the migratory locust, Locusta migratoria

ZHAO Xiao-ming, YANG Jia-peng, GOU Xin, LIU Wei-min, ZHANG Jian-zhen

Journal of Integrative Agriculture 2021, 20 (6): 1596-1606. DOI: 10.1016/S2095-3119(20)63248-8

摘要（115）

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表皮蛋白是昆虫表皮关联器官的主要组成部分，如体壁和翅，然而其在渐变态昆虫翅发育中的功能尚不清楚。本文从飞蝗中鉴定出一种属于CPR 家族RR-2亚家族的翅表皮蛋白LmACP8。LmACP8主要在翅芽中表达，且在三龄、四龄和五龄若虫蜕皮前表达量较高，其编码的蛋白定位于翅芽和成虫翅的原表皮层。利用RNA干扰，LmACP8的表达缺失显著降低了其蛋白质含量，从而导致飞蝗若虫向成虫转变过程中翅形态发生异常。进一步研究证实这种异常形态发生是由于翅内皮层严重受损所致。个体实验结果发现，蜕皮激素（20-hydroxyecdysone，20E）能够抑制LmACP8的表达，而在干扰激素受体基因LmHR39（Hormone receptor 39）后，LmACP8表达显著上调。由此可见，LmACP8参与飞蝗若虫向成虫转变过程中翅的发育，且其表达受LmHR39介导的20E信号通路负调控。

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8. Molecular characteristics and temperature tolerance function of the transient receptor potential in the native Bemisia tabaci AsiaII3 cryptic species

JI Shun-xia, SHEN Xiao-na, LIANG Lin, WANG Xiao-di, LIU Wan-xue, WAN Fang-hao, Lü Zhi-chuang

Journal of Integrative Agriculture 2020, 19 (11): 2746-2757. DOI: 10.1016/S2095-3119(20)63226-9

摘要（111）

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Insects are poikilothermic animals, and temperature is one of the most important abiotic factors affecting their spread and distribution. For example, differences in thermal tolerance may underlie the significant differences in geographical distributions between the native AsiaII3 and invasive MED (Mediterranean) cryptic Bemisia tabaci species in China. Transient receptor potential (TRP) channels are key components of the insect temperature perception system and act as molecular thermometers since they can be activated by specific changes in temperature. In this study, we cloned and characterized the AsiaII3 BtTRP gene and revealed its functions in the response to thermal stress. The full-length cDNA of BtTRP was 3 821 bp, with a 3 501-bp open reading frame encoding a 132.05-kDa protein. Comparing the deduced amino acid sequences of AsiaII3 BtTRP and MED TRP revealed five amino acid differences. In situ hybridization indicated that BtTRP might be widely expressed throughout the AsiaII3 adult body. BtTRP mRNA expression reached the highest levels after exposure to mild thermal stimuli (12 and 35°C), showing that BtTRP expression can be induced by temperature stress. Furthermore, the thermal tolerance of AsiaII3 after BtTRP dsRNA feeding was significantly lower than that of the control. Taken together, the present study highlights the importance of TRP channels for B.?tabaci thermal resistance, and allows us to infer that the differences in amino acids between AsiaII3 and MED might cause the differences in thermal tolerance of these two cryptic species. This study provides a new direction for investigating geographic distribution differences between invasive and native insects.

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9. Cloning and functional characterization of two peptidoglycan recognition protein isoforms (PGRP-LC) in Bactrocera dorsalis (Diptera: Tephritidae)

WEI Dong, WANG Zhe, XU Hui-qian, NIU Jin-zhi, WANG Jin-jun

Journal of Integrative Agriculture 2020, 19 (12): 3025-3034. DOI: 10.1016/S2095-3119(20)63202-6

摘要（85）

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The innate immune system of insects is the front line of self-defense against pathogen invasion. Peptidoglycan recognition proteins (PGRPs) are important components and play key roles in insect immune systems by recognizing peptidoglycan (PGN) in bacterial cell walls. We characterized two isoforms of the PGRP-LC gene, BdPGRP-LCa and BdPGRP-LCb, from Bactrocera dorsalis (Hendel), an important fruit and vegetable pest worldwide. These two isoforms contain an open reading frames of 1 668 bp and 1 731 bp, encoding a protein of 555 and 576 amino acids, respectively. Quantitative real-time PCR results showed that both transcripts were prominently expressed in midgut and fat body of B. dorsalis adult. Inoculation of pathogens showed that both isoforms actively responded to Escherichia coli PGN. We also observed a light response to Staphylococcus aureus PGN. Upon Beauveria bassiana inoculation, the expression of BdPGRP-LCa was enhanced, but the expression of BdPGRP-LCb was suppressed. Suppression of both transcripts by RNA interference led to increased mortality of flies challenged by E. coli, indicating that the two isoforms are involved in sensing Gram-negative bacterial infections.

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