Molecular Cloning and Functional Analysis of UDP N-acetylglucosamine Pyrophosphorylases Gene in Frankliniella occidentalis
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摘要:目的 阐明几丁质合成通路中的关键基因UDP-N-乙酰氨基葡萄糖焦磷酸化酶(UDP N-acetylglucosamine pyrophosphorylases, UAP)对西花蓟马Frankliniella occidentalis生长发育的影响。方法 基于西花蓟马转录组数据，克隆验证了UAP基因的开放阅读框(Open Reading Frame, ORF)全长序列，命名为FoccUAP。利用RT-qPCR方法分析FoccUAP在各个组织和不同龄期的表达情况。最后通过向西花蓟马蜕皮24 h的2龄若虫显微注射dsUAP，检测沉默效率，观察其对西花蓟马生长发育的影响。结果 FoccUAP基因开放阅读框全长1 545 bp，编码514个氨基酸，预测蛋白分子量为56.738 kDa，具有Glyco - tranf - GTA - type Super family家族的保守结构域，与等翅目、蜚蠊目、直翅目和半翅目昆虫的UAP基因亲缘关系较近。FoccUAP基因在西花蓟马各个组织都有表达，在头部和腹部相对表达量最高，触角、胸部和足相对表达量较低。该基因在虫体生长发育的各个阶段均有表达，其中2龄幼虫、蛹以及羽化后24、48、168、240 h的成虫表达量分别是1龄幼虫的1.30、2.50、1.56、2.0、2.43和2.56倍。RNAi结果表明，注射dsUAP的西花蓟马羽化率（36.3%）和注射后120 h的存活率（5.0%）均显著低于dsGFP对照组，且注射dsUAP的西花蓟马成虫出现翅膀发育不完整、胸腹部皱缩不规则等畸形现象。结论 FoccUAP基因对西花蓟马生长发育具有重要的作用。
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关键词:
- 西花蓟马 /
- UDP-N-乙酰氨基葡萄糖焦磷酸化酶 /
- 几丁质合成 /
- RNAi
Abstract:Objective Effects of UDP N-acetylglucosamine pyrophosphorylases (UAP) on the growth and development of Frankliniella occidentalis were studied.Method Based on a transcriptome dataset, an open reading frame (ORF) of UAP gene of F. occidentalis (FoccUAP) was cloned. Expressions of the FoccUAP in different body tissues and growth stages of the insect were detected by RT-qPCR. Effects of UAP gene on the growth and development were examined by microinjecting dsRAN in the 2nd instars (eclosion about 24 h) of F. occidentalis.Result The full-length of FoccUAP ORF was 1 545 bp and encoded 514 amino acids with a predicted molecular mass of 56.738 kDa. FoccUAP contained the conserved domain of glyco-tranf-GTA-type super family, which had a close evolutionary relationship with the orthologues in Isoptera, Blattaria, Orthoptera, and Hemiptera. The RT-qPCR results showed that FoccUAP was highly expressed in the head and abdomen, detected in the antenna, thorax and leg, and found in all developmental stages of the insect. The relative transcript levels of FoccUAP in the 2nd instar, pupa, adult-24 h, adult-48 h, adult-168 h, and adult-240 h were 1.30-, 2.50-, 1.56-, 2.0-, 2.43-, and 2.56-fold, respectively, of those in the 1st instar. Further examination on the RNAi showed that the injection of dsUAP into the 2nd instar F. occidentalis significantly decreased the eclosion rate (36.3%) and the survival rate (5.0% in 120 h after microinjection) as compared to dsGFP controls. Moreover, the dsUAP treatment also induced abnormal phenotypes on wings and abdomen–thorax of the thrips.Conclusion UAP played a significant role in the growth and development of F. occidentalis. -
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图 1 FoccUAP基因的核苷酸及预测的氨基酸序列
注：下划线部分为信号肽氨基酸序列；双下划线为蛋白保守结构域氨基酸序列；*为终止密码子。
Figure 1. Nucleotide and deduced amino acid sequence of FoccUAP
Note: The signal peptide sequence (MEHQYENLRN) is marked with underline. The conserved amino acid sequences of Glyco-tranf-GTA-type superfamily are marked with double underline. The TAA stop codon is marked with *.
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图 3 FoccUAP基因在西花蓟马不同发育时期（A）和不同组织（B）的mRNA表达分析
注：数据上不同的字母表示差异显著（Tukey HSD test, P＜0.05）
Figure 3. Relative expression levels of FoccUAP mRNA in different developmental stages (A) and tissues (B) of F. occidentalis
Note: Different lowercase letters means significant difference (Tukey HSD test, P＜0.05).
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图 4 注射dsUAP对西花蓟马死亡率及生长发育的影响
注：*或者不同的字母表示差异显著（Tukey HSD test, P＜0.05）
Figure 4. Effects of dsUAP on growth and mortality rate of F. occidentalis
Note：* or different lowercase letters means significant difference (Tukey HSD test, P＜0.05).
表 1 引物信息
Table 1 Primers used
引物名称
Primer name序列
SequenceFoccUAP-ORF-F ATGGAGCACCAGTACGAAAACCTC FoccUAP-ORF-R TTAGTGAACACCATTTGATATCCCA dsFoccUAP-F TAATACGACTCACTATAGGGTACAAATGCTACCTGCACTGAAAGTAAGCT dsFoccUAP-R TAATACGACTCACTATAGGGTGGCAACATCATCCTGGATTCTCC qFoccUAP-F TCCGTTTACTGATAGTCTTGTTGTCTGGG qFoccUAP-R GCTGTGCGTGGGCAATCCTTTT qβ-actin-F CACCACCGCTGAGCGTGAAATCG qβ-actin-R GTGATGACCTGACCGTCGGGAAGC dsGFP-F TAATACGACTCACTATAGGGCGAGGAGCTGTTCACCGG dsGFP-R TAATACGACTCACTATAGGGTCCTCGATGTTGTGGCGG 
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