Genomic and functional analysis of gene CpMYB24 in papaya

Zhang, Xiaodan

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https://hdl.handle.net/2142/115921 Copy

Description

Title

Genomic and functional analysis of gene CpMYB24 in papaya

Author(s)

Zhang, Xiaodan

Issue Date

2022-07-13

Director of Research (if dissertation) or Advisor (if thesis)

Ming, Ray

Doctoral Committee Chair(s)

Ming, Ray

Committee Member(s)

• Tranel, Patrick
• Marshall-Colon, Amy
• Jamann, Tiffany

Department of Study

Plant Biology

Discipline

Plant Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Functional study
• Stamen development
• Papaya

Abstract

Papaya (Carica papaya L.) is a major fruit crop in tropical and subtropical regions. It is a trioecious species with three sex types: female, male, and hermaphrodite. Sex in papaya is controlled by a pair of sex chromosomes with two slightly different Y chromosomes: Y determines males and Yh determines hermaphrodites, but the mechanisms of papaya sex determination remain unclear. A gene, CpMYB24, located in the 1 Mb deletion of male-specific region of the Y chromosome (MSY) in a papaya male-to-female sex reversal mutant, is a strong candidate male activator in papaya. CpMYB24 was present on X, Y, and Yh chromosomes, and the orthologous coding sequences of Y and Yh alleles were identical, while CpMYB24_X and CpMYB24_Y alleles were slightly different in coding sequences. CpMYB24 was highly expressed in male and hermaphrodite flowers but not in female flowers, indicating its role in male reproductive development. Upon overexpressing each CpMYB24 allele in Arabidopsis double mutant myb21 myb24, only the CpMYB24_Y allele partially rescued the male sterility phenotype, which indicated that CpMYB24_Y retained the conserved role in male development whereas the X allele lost this function. To verify the role of CpMYB24 as a male activator in papaya, CpMYB24 was knocked out via the CRISPR/cas9 gene-editing system. Unexpectedly, the knockout of CpMYB24 inhibited root development and caused a high lethality rate of transgenic seedlings. RNA-seq analysis was performed to investigate the causes of phenotypes after the knockout of CpMYB24 in transgenic plants. The RNA-seq results revealed the role of CpMYB24 in the metabolic pathways, biosynthesis of secondary metabolites, and hormone signaling transduction. Root development has been reported to be regulated by plant hormones and their interactions. CpMYB24 is most likely involved in the auxin signaling pathway, which further regulates root development in papaya. A high-quality yeast two-hybrid (Y2H) library was constructed for identifying the proteins that interact with CpMYB24_Y. Ten proteins that interact with CpMYB24 were identified, including two transcription factors, CpMYC2 and CpTCP20. Bimolecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI) assays further confirmed the interactions between CpMYB24_Y and both CpMYC2 and CpTCP20. DNA-affinity-purified sequencing (DAP-seq) was conducted to identify target genes of both alleles of CpMYB24. The two proteins collectively bind to 557 genes, 252 of which are unique to CpMYB24_X and 102 are unique to CpMYB24_Y. Unique target genes of CpMYB24_Y were strong downstream candidates for involvement in stamen development since only the Y allele regulates this process. The results showed that CpMYB24_Y likely regulates papaya stamen development by mediating through jasmonate and ABA signaling pathways. Combining DAP-seq data with RNA-seq data of CpMYB24 knockout transgenic papaya identified an auxin-related SAUR gene, indicating that CpMYB24 regulates a SAUR gene involved in the auxin signaling pathway to control root development. Our work characterized the function of CpMYB24 in hormone-mediated stamen development and root development and revealed molecular regulatory networks underlying papaya developmental processes.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Xiaodan Zhang

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