fl(2)d单等位基因的敲除显著降低小菜蛾的生殖力和育性

doi:10.3864/j.issn.0578-1752.2021.14.009

Abstract

Abstract:

【Objective】 RNA methylation is the main form of epigenetic modification at post-transcriptional level, which is involved in many important cellular processes. The diamondback moth, Plutella xylostella, is an important oligophagous insect pest, causing serious loss on the production of cruciferous vegetables. However, the function of RNA methylation-related genes in P. xylostella is still unclear. The present study aims to identify and clone the homologous fl(2)d, one of the members of the RNA methylation protein complex (writers), to determine the expression pattern offl(2)d, and to knockout fl(2)d using CRISPR/Cas9 for the investigation of its biological functions in P. xylostella.【Method】The sequence of homologous fl(2)d was identified in the genome database ofP. xylostella, which was used for PCR amplification of the coding sequence (CDS). Quantitative real-time PCR (qRT-PCR) was used to study the relative expression levels of fl(2)d in different developmental stages and adult gonads of P. xylostella. The fl(2)d was edited using CRISPR/Cas9 combined with egg injection. Each of the adults that developed from the injected eggs was used to pair with a wild-type adult for reproduction. Offspring of the same population was forced to inbreed by single-pair mating to establish the mutant strains. The differences of genetic characters, biological parameters and phenotypes between mutants and wild-type individuals were recorded and compared to decipher the function offl(2)d.【Result】The CDS of fl(2)d with length of 912 bp was isolated, the expression of which was high in female pupa, adult and egg, moderate in male adult and pupa, the lowest in larva, and significantly higher in ovary than in testis of adult. The sgRNAs targeting fl(2)d and the Cas9 protein were mixed to inject eggs, and the offsprings carrying mutant alleles were screened for homozygous strains based on single-pair inbreeding for 10 generations. Three types of heterozygous mutant strains both predicted to cause frameshift of the CDS were obtained, with the deletion of 4 (Δfl(2)d213-4), 5 (Δfl(2)d213-5) and 7 (Δfl(2)d213-7) bases. During the screening process, six and two homozygous mutants from Δ fl(2)d213-4 and Δ fl(2)d213-5 strains were identified, respectively. The homozygous mutants of Δ fl(2)d213-4 successfully mated in two pairs, but no eggs were produced. Meanwhile, each two male adults of homozygous mutants of either Δ fl(2)d213-4 or Δ fl(2)d213-5 were mated with the same type of female heterozygous mutant, and also no eggs were produced. The results indicated that individuals with homozygousfl(2)d mutation may have extremely low survival rate and not be able to produce offspring. Through analyzing separation ratio of the genotypes of offspring from the inbreeding of heterozygous mutants and the hybridization between heterozygous mutants and wild-type, it was found that the ratio of heterozygous mutant individuals to wild-type was slightly less than 2 and 1, respectively, indicating that heterozygous mutation of fl(2)d would affect the normal growth and development of P. xylostella, and in some cases would lead to death. The offsprings of mutant individuals, which carry a mutant allele, showed a sex ratio close to 1﹕1 (P<0.05). It was speculated that thefl(2)d might not be involved in sex determination in P. xylostella. For the mating consists of mutant adults, the fecundity and hatchability were significantly lower (P<0.01) than the mating between wild-type adults. Most of the eggs produced from the mutant parents look abnormal, and could not hatch normally due to water loss and shrinkage. Based on the dissection of adult gonads, it was found that the number of attached eggs on the ovary of the mutant female adult and the wild-type female adult that has mated with mutant male adult was less than that of the wild-type virgin female adult, while no obvious abnormality was found for the testis of mutant male adult. Some of the hatched heterozygous mutants showed different degrees of distortion during the whole developmental process, resulting in the failure to complete life cycle. A small part of the heterozygous mutant individuals could develop normally, and thus transmit the mutant allele to their offspring. According to our findings, a model of genetic control ofP. xylostella based on fl(2)d was proposed.【Conclusion】The fl(2)d is involved in the reproductive process and embryonic development ofP. xylostella, mutation of which significantly affects the population size of the offspring, making it an ideal target for the genetic control of P. xylostella.

Key words: Plutella xylostella, fl(2)d, heterozygous mutant, fecundity, fertility

LI FeiFei,WANG BeiBei,LAI YingFang,YANG FeiYing,YOU MinSheng,HE WeiYi. Knockout of Single Allele of fl(2)d Significantly Decreases the Fecundity and Fertility inPlutella xylostella[J].Scientia Agricultura Sinica, 2021, 54(14): 3029-3042.

Figures/Tables 8

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引物名称Primer name	序列Sequence (5′-3′)	用途Usage
Fl(2)d-ORF-F	CGAGGAGACAGAACGCCTT	克隆fl(2)d Cloning offl(2)d
Fl(2)d-ORF-R	GGTGACGGTGAGGGGTTC	克隆fl(2)d Cloning offl(2)d
qPCR-Fl(2)d-F	CGGAGCTCAAATCATCACACGC	fl(2)d定量表达 qRT-PCR of fl(2)d
qPCR-Fl(2)d-R	GCCCAGGTCCTCATTCTCCT	fl(2)d定量表达 qRT-PCR of fl(2)d
RPL32-F	CAATCAGGCCAATTTACCGC	内参基因RPL32定量表达 qRT-PCR of reference RPL32
RPL32-R	CTGGGTTTACGCCAGTTACG	内参基因RPL32定量表达 qRT-PCR of reference RPL32
Fl(2)d-Mut-TF	GTATAATCCAATGAAGGTATGACAG	突变检测 Mutation detection
Fl(2)d-Mut-TR	CCTACAGTGAAACCCGCAA	突变检测 Mutation detection
CRISPR-F1	TAATACGACTCACTATAGGAAGCCTGGAAAAGGCGAAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC	合成sgRNA Synthesis of sgRNAs
CRISPR-F2	TAATACGACTCACTATAGGAAGGCTAGCAGCTAAAGAACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC
CRISPR-R	AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAA

G2代至G10代不同基因型个体数 Numbers of different genotypes from G2 to G10	AA×Aa（亲本基因型Parental genotype）								Aa×Aa（亲本基因型Parental genotype）
	Δfl(2)d-4		Δfl(2)d-5		Δfl(2)d-7		P=0.05, df=1, χ²=3.841		Δfl(2)d-4			Δfl(2)d-5			Δfl(2)d-7			P=0.05, df=1, χ²=3.841
	AA	Aa	AA	Aa	AA	Aa	Aa/AA	χ²	AA	Aa	aa	AA	Aa	aa	AA	Aa	aa	Aa/AA	χ²
G2	13	13	11	11	22	20	0.957	0.044	6	16	0	/	/	/	3	9	0	2.778	0.721
G3	19	19	/	/	40	30	0.831	0.926	15	24	3	7	5	2	10	16	0	1.406	2.506
G4	/	/	/	/	14	14	1.000	0	7	16	3	14	30	0	11	19	0	2.031	0.095
G5	20	14	25	37	1	3	1.174	0.640	/	/	/	11	17	0	13	23	0	1.667	0.500
G6	5	3	23	21	20	8	0.667	3.200	/	/	/	15	25	0	2	6	0	1.824	0.094
G7	17	7	18	12	19	15	0.630	4.545	/	/	/	2	10	0	/	/	/	5.000	2.063
G8	6	6	6	4	19	15	0.806	0.643	/	/	/	3	7	0	1	3	0	2.500	0.339
G9	/	/	7	9	11	9	1.000	0	/	/	/	/	/	/	/	/	/	/	/
G10	/	/	/	/	18	26	1.444	1.455	/	/	/	3	5	0	/	/	/	1.667	0.031
合计Total	80	62	90	94	164	140	0.886	0.292	28	56	6	55	99	2	40	76	0	1.878	0.488

Knockout of Single Allele of fl(2)d Significantly Decreases the Fecundity and Fertility inPlutella xylostella

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