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This study explored the key B genes and their nucleic acid and protein sequence characteristics of the stamens of green pepper.
The expression patterns of the flowers in the process of male and female flower differentiation were analyzed, so as to provide candidate genes and data support for the genetic regulation mechanism of the male and female flower bud differentiation of green peppercorns.
Based on the transcriptome and genome database of green peppercorns, all the differentially expressed B MADS-box family genes during the differentiation of male and female flowers were extracted, and the key members were analyzed.
The coding sequences of key member genes were cloned to comprehensively analyze the characteristics of nucleic acid and protein sequences, and the expression patterns of key member genes were analyzed, and their expression patterns in 11 different tissues and organs and the differentiation process of male and female flowers were analyzed.
Genes and genetic regulation mechanisms of green peppercorns
Green peppercorns, also known as bamboo-leaved peppercorns, are small perennial trees or shrubs in the family Rutaceae. The common green peppercorns are mainly pure female plants, with clustered umbel-shaped panicles, and the lack of petals in the unisexual incomplete flowers, and the main reproduction mode is non-fusion reproduction.
It is an important economic tree species for both spices, oilseeds, and pharmaceuticals, and is one of the pillar industries of rural revitalization in the western part of the mainland, with high research and economic value.
However, in recent years, field observations have found that a large number of male flowers appear in green peppercorns, and there is a trend of increasing year by year, which leads to a significant decrease in the fruit bearing yield of green peppercorns propagated by parthenogenesis, which seriously affects its industrial promotion and application.
Therefore, exploring the key regulatory genes of the male flower bud of Zanthoxylum bungeanum is an important premise for revealing the genetic regulatory mechanism of the male flower differentiation of Zanthoxylum bungeanum and analyzing the internal reasons for the formation of male flowers of Zanthoxylum bungeanum bungeanum flowers.
It is of great significance for the research and development of gender regulation technology for male flower prevention and control and the cultivation of high-quality and high-yield green pepper germplasm resources.
Previous studies have shown that the structure of angiosperm flowers is generally conserved, which is composed of sepals, petals, stamens and carpels from the outside to the inside, and is controlled by the combination of A, B and C3 functional genes.
Most of the genes in the ABC model of flower organ differentiation belong to the MADS family members, and the proteins encoded by them are mainly composed of four domains with different degrees of conservatism: MADS-box, I-spacer, K-box and C-terminal, so they are also called MIKC-type transcription factors.
AMONG THEM, CLASS B GENES ARE MEMBERS OF THE APETALA3/PISTILLATA SUBFAMILY, WHICH ARE MAINLY INVOLVED IN THE DIFFERENTIATION PROCESS OF STAMENS AND CAN SYNERGISTICALLY REGULATE THE DEVELOPMENT OF PETALS WITH CLASS A GENES.
A number of studies have reported that B genes play an important role in regulating stamen differentiation in different types of plants such as flowers, vegetables, fruits, and woody plants.
However, although PI homologous genes have been thoroughly studied in some species, the number of PI genes and their regulatory effects vary among different species.
Expert studies have shown that three PI/AP3 family members have been identified in Arabidopsis, among which pi-1 has the most obvious regulatory effect. Similarly, among the seven PI/AP3 family members of Sycopodium sylvestris, HoPI_3 significantly regulated the differentiation and formation of male inflorescences.
At present, the key regulatory genes and genetic regulatory mechanisms in the process of sexual differentiation of green pepper flower buds have not been reported.
Therefore, it is of great significance to explore the important regulatory genes and genetic information of PI/AP3 family in P. zanthoxylum bungeanum to reveal the molecular mechanism of stamen formation.
Combined with the transcriptome database of the male and female flower differentiation process of green pepper in the early stage of the research group, the important PI genes regulating the differentiation of stamens of green pepper were screened and obtained.
Combined with genomic data, the full length of the coding sequence of the gene was successfully cloned, the nucleic acid and protein sequence characteristics of ZaPI were analyzed, and the transcription factors and cis-acting elements in response to ZaPI were analyzed.
At the same time, combined with transcriptome data and qRT-PCR technology, the expression patterns of ZaPI gene in 11 different tissues and organs such as roots, stems, leaves, and spines of green peppercorn, as well as 10 time points of male and female flower differentiation were comprehensively clarified.
The results of this study provide a rich theoretical basis and data support for exploring the molecular mechanism of stamens formation and developing the sex regulation technology for male flower prevention and control.
Analysis of ZaPI gene expression pattern in green peppercorn
In order to comprehensively reveal the tissue-specific expression pattern of ZaPI gene in green pepper, the expression abundance of ZaPI in five samples including roots, stems, mature leaves, fruits and leaf buds was extracted from the transcriptome dataset in the previous stage of the research group.
At the same time, the expression abundance of ZaPI in 6 samples of young flowers, young leaves, peels, seeds, terminal buds and skin spines was analyzed from the transcriptome data published by NCBI, and the RNA-seq data of all samples had 3 biological replicates.
At the same time, 10 differentiation time points samples of male and female flowers of green pepper were collected, and the RNA of each sample was extracted by RNA rapid extraction kit, and cDNA was obtained by reverse transcription.
According to the ZaPI coding sequence obtained in this study, the PrimerPremier5 was used to design specific quantitative detection primers, and qRT-PCR was used to detect the expression pattern of ZaPI gene in each sample.
The relative expression level of ZaPI gene in different samples was calculated by 2-ΔΔCt method, and the difference in the relative expression of PI between different samples was analyzed by Duncan multiple comparison, and the significance level was P<0.05, and GraphPadPrism5 was used to draw a histogram.
A total of 11 AP3/PI family members were obtained from the differentially expressed gene set of male and female flowers of P. zanthoxylum, including 3 ZaPI genes and 8 ZaAP3 genes.
The analysis of gene expression pattern showed that the differentially obtained genes were significantly up-regulated at the maturity stage of stamens, but the expression level was low during the differentiation of female flowers.
At the same time, the expression level of Zardc17043 in the male flower M4 stage was 15-90 times higher than that of other differential genes, and the expression of Zardc17043 was significantly up-regulated during the male flower differentiation process.
These results suggest that Zardc17043 may be an important member of the AP3/PI family involved in the regulation of male flowers of Prickoxylum bungeanum chinensis. Therefore, this gene was selected as the key regulatory gene for stamens differentiation in green peppercorns for follow-up analysis and research.
Through the comparison analysis of PI genes with other species, it was found that the coding sequence of Zardc17043 obtained from the genome database of green pepper was only 426 bp in length, and the C-terminal protein domain was missing.
However, the predicted coding sequence length of the homologous gene Cluster-12235.70400 obtained from the non-reference transcriptome database of different tissues was 769 bp, which had high homology with Zardc17043, but lacked the start codon of protein translation.
Therefore, in this study, a coding sequence with a length of 624 bp was successfully obtained, with the start codon being ATG and the stop codon being TGA, encoding a total of 207 amino acids, which was preliminarily determined to be the full length of the coding sequence of Zardc17043 in green pepper.
In order to confirm the accuracy of the above analysis results, the cDNA of the male flowers of green pepper was used as a template for multiple PCR amplifications.
The results showed that except for an amino acid mutation at the N-terminus of the sequence, the other PCR amplification sequences were completely consistent with the sequences obtained by the above splicing.
In addition, the results of PI protein sequence alignment of different species showed that ZaPI protein contained typical MADS-box and K-box domains, and had relatively intact C-terminal transcriptional activation regions.
Transcription factor binding site of ZaPI in green peppercorns
In order to better understand the potential function and regulatory genes of ZaPI gene in green pepper, PlantRegMap software was used to predict it.
A total of 54 binding sites belonging to 17 transcription factor families, including BR-BPC, MIKC_MADS, C2H2 and MYB, were obtained, among which BR-BPC had the largest number of binding sites, with 10 sites (19%), followed by MIKC_MADS, with 9 sites (17%).
These transcription factors were significantly related to the regulation of plant reproductive growth, physiological metabolism, cell differentiation and other growth and development processes, suggesting that ZaPI may interact with these transcription factors to regulate the differentiation process of green pepper stamens.
In addition to the characteristic elements such as CAAT-box and TATA-box in the promoter itself, there are also six types of response elements, which are 9% hormone response elements, 8% light response elements, 7% MYB response elements, 3% anaerobic inducible regulatory elements, 2% defense and stress response elements, and meristem expression response elements.
These results suggest that ZaPI gene may have multiple signal response modes, which are involved in different life processes of green peppercorns, and are potential plant hormones and light signal responders.
It may be regulated by the interaction of MYB transcription factors, and plays an important role in regulating plant reproductive growth under stress conditions.
The results of tissue-specific analysis showed that ZaPI was almost not expressed in 10 different tissues and organs, such as rhizomes and leaf spines, and the expression level was relatively high in fruits.
In addition, in order to further clarify the expression of ZaPI gene in the sexual differentiation process of green peppercorns, qRT-PCR detection and analysis were carried out using 10 time point samples of female and male stamens differentiation.
The results showed that there was a significant difference in the ZaPI gene in the process of male and female flower differentiation. ZaPI was mainly expressed in male flowers, and the expression level was gradually up-regulated with the differentiation of stamens.
The expression level of stamen formation in December and stamen maturity in February and March was significantly increased, especially the expression of the latter was exponentially up-regulated. However, the expression level of ZaPI in the whole female flower differentiation process of green pepper remained at a relatively low level.
The above results were consistent with the basic functions of class B genes, indicating that the ZaPI gene obtained in this study played an important regulatory role in the differentiation of male flowers of Zanthoxylum bungeanum chinensis.
Combined with the transcriptome and genomic data of green peppercorns, a complete coding sequence of ZaPI gene with a length of 624 bp was successfully cloned.
Protein domain analysis, phylogenetic tree construction, chromosome mapping, transcription factor binding sites and cis-acting element prediction were used for comprehensive bioinformatics analysis.
It was found that ZaPI gene may regulate the differentiation process of flower organs in green pepper in response to signaling pathways such as plant hormones, light and stress stress.
In addition, transcriptomics and real-time PCR analysis showed that the gene was almost not expressed in the vegetative organs of green pepper, and the expression level of female flowers during the differentiation process was relatively low.
However, the expression level was significantly up-regulated during the differentiation of male flowers, suggesting that it may be significantly involved in the genetic regulation process of stamens differentiation of green peppercorns.
The results obtained in this study provide rich theoretical support for further exploring the molecular mechanism of stamens formation and cultivating high-yield green pepper germplasm resources