[Identification and functional analysis of jasmonic acid signaling repressor McJAZ8 gene in Mentha canadensis].

Mentha canadensis, as a plant with medicinal and culinary uses, holds significant economic value. Jasmonic acid signaling repressor JAZ protein has a crucial role in regulating plant response to adversity stresses. The M. canadensis McJAZ8 gene is cloned and analyzed for protein characterization, protein interactions, and expression patterns, so as to provide genetic resources for molecular breeding of M. canadensis for stress tolerance. This experiment will analyze the protein structural characteristics, subcellular localization, protein interactions, and gene expression of McJAZ8 using bioinformatics, yeast two-hybrid(Y2H), transient expression in tobacco leaves, qRT-PCR, and other technologies. The results show that:(1)The full length of the McJAZ8 gene is 543 bp, encoding 180 amino acids. The McJAZ8 protein contains conserved TIFY and Jas domains and exhibits high homology with Arabidopsis thaliana AtJAZ1 and AtJAZ2.(2)The McJAZ8 protein is localized in the nucleus and cytoplasm.(3)The Y2H results show that McJAZ8 interacts with itself or McJAZ1/3/4/5 proteins to form homologous or heterologous dimers.(4)McJAZ8 is expressed in different tissue, with the highest expression level in young leaves. In terms of leaf sequence, McJAZ8 shows the highest expression level in the fourth leaf and the lowest expression level in the second leaf.(5) In leaves and roots, the expression of McJAZ8 is upregulated to varying degrees under methyl jasmonate(MeJA), drought, and NaCl treatments. The expression of McJAZ8 shows an initial upregulation followed by a downregulation pattern under CdCl_2 treatment. In leaves, the expression of McJAZ8 tends to gradually decrease under CuCl_2 treatment, while in roots, it initially decreases and then increases before decreasing again. In both leaves and roots, the expression of McJAZ8 is downregulated to varying degrees under AlCl_(3 )treatment. This study has enriched the research on jasmonic acid signaling repressor JAZ genes in M. canadensis and provided genetic resources for the molecular breeding of M. canadensis.

[Cloning and expression pattern analysis of abscisic acid receptor gene McPYL4 in Mentha canadensis].

Mentha canadensis is a traditional Chinese herb with great medicinal and economic value. Abscisic acid(ABA) receptor PYLs have important roles in plant growth and development and response to adversity. The M. canadensis McPYL4 gene was cloned, and its protein characteristics, gene expression, and protein interactions were analyzed, so as to provide genetic resources for genetic improvement and molecular design breeding for M. canadensis resistance. Therefore, the protein characteristics, subcellular localization, gene expression pattern, and protein interactions of McPYL4 were analyzed by bioinformatics analysis, transient expression of tobacco leaves, RT-qPCR, and yeast two-hybrid(Y2H) techniques. The results showed that the McPYL4 gene was 621 bp in length, encoding 206 amino acids, and its protein had the conserved structural domain of SRPBCC and was highly homologous with Salvia miltiorrhiza SmPYL4. McPYL4 protein was localized to the cell membrane and nucleus. The McPYL4 gene was expressed in all tissue of M. canadensis, with the highest expression in roots, followed by leaves, and it showed a pattern of up-regulation followed by down-regulation in leaves 1-8. In both leaves and roots, the McPYL4 gene responded to the exogenous hormones ABA, MeJA, and the treatments of drought, AlCl_3, NaCl, CdCl_2, and CuCl_2. Moreover, McPYL4 was up-regulated for expression in both leaves and roots under the MeJA treatment, as well as in leaves treated with AlCl_3 stress for 1 h, whereas McPYL4 showed a tendency to be down-regulated in both leaves and roots under other treatments. Protein interactions showed that McPYL4 interacted with AtABI proteins in an ABA-independent manner. This study demonstrated that McPYL4 responded to ABA, JA, and several abiotic stress treatments, and McPYL4 was involved in ABA signaling in M. canadensis and thus in the regulation of leaf development and various abiotic stresses in M. canadensis.

Transcriptome-Wide Identification and Characterization of the JAZ Gene Family in Mentha canadensis L.

Jasmonate ZIM-domain (JAZ) proteins are the crucial transcriptional repressors in the jasmonic acid (JA) signaling process, and they play pervasive roles in plant development, defense, and plant specialized metabolism. Although numerous JAZ gene families have been discovered across several plants, our knowledge about the JAZ gene family remains limited in the economically and medicinally important Chinese herb Mentha canadensis L. Here, seven non-redundant JAZ genes named McJAZ1-McJAZ7 were identified from our reported M. canadensis transcriptome data. Structural, amino acid composition, and phylogenetic analysis showed that seven McJAZ proteins contained the typical zinc-finger inflorescence meristem (ZIM) domain and JA-associated (Jas) domain as conserved as those in other plants, and they were clustered into four groups (A-D) and distributed into five subgroups (A1, A2, B1, B2, and D). Quantitative real-time PCR (qRT-PCR) analysis showed that seven McJAZ genes displayed differential expression patterns in M. canadensis tissues, and preferentially expressed in flowers. Furthermore, the McJAZ genes expression was differentially induced after Methyl jasmonate (MeJA) treatment, and their transcripts were variable and up- or down-regulated under abscisic acid (ABA), drought, and salt treatments. Subcellular localization analysis revealed that McJAZ proteins are localized in the nucleus or cytoplasm. Yeast two-hybrid (Y2H) assays demonstrated that McJAZ1-5 interacted with McCOI1a, a homolog of Arabidopsis JA receptor AtCOI1, in a coronatine-dependent manner, and most of McJAZ proteins could also form homo- or heterodimers. This present study provides valuable basis for functional analysis and exploitation of the potential candidate McJAZ genes for developing efficient strategies for genetic improvement of M. canadensis.

Novel inactivating mutations in the FSH receptor cause premature ovarian insufficiency with resistant ovary syndrome.

RESEARCH QUESTION: What is the genetic aetiology of three resistant ovary syndrome (ROS) pedigrees from 13 Chinese Han families with non-syndromic premature ovarian insufficiency (POI). DESIGN: The proband in each family was subjected to whole-exome sequencing. Bioinformatic and in-vitro functional analyses were performed for the functional characterization of the FSHR mutations. RESULTS: Four novel mutations, two homozygous mutations (c.419delA, c.1510C>T), and a compound heterozygous mutation (c.44G>A and deletion of exons 1 and 2) of FSHR were identified in the three non-syndromic POI-with-ROS families. Bioinformatic analysis predicted that the three novel point mutations in FSHR are deleterious and associated with POI in the three families, which was confirmed by in-vitro functional analysis, in which FSH-induced adenosine 3',5'-cyclic monophosphate production was abolished for all receptors. CONCLUSIONS: The three novel point mutations in FSHR were all functional inactivating mutations, and were the genetic aetiology of the three non-syndromic POI-with-ROS families. The first FSHR frameshift mutation is reported here, and the first missense mutation in the signal peptide-encoding region of FSHR to be associated with POI. Women affected by ROS should consider undergoing mutation screening for FSHR.

[Identification of novel KIT gene mutations in two Chinese families with piebaldism].

OBJECTIVE: To screen for potential mutations of KIT gene for two Chinese families affected with piebaldism in order to facilitate genetic counseling and assisted reproduction. METHODS: Peripheral blood samples were collected from 2 patients of family 1 and the proband and 3 unaffected members of family 2 for the extraction of DNA and RNA. PCR-sequencing and reverse transcription PCR-sequencing were used to screen KIT mutations. RESULTS: All of the patients from family 1 were found to carry heterozygous IVS12+2-+7delinsACATCTTTA, a splicing mutation undocumented in the human gene mutation data base (HGMD) database. This mutation has resulted in c.1765-1779del in cDNA and p.Gly592Ala/del:E12, which has led to skipping of exon 12 and no expression of cDNA. The proband from family 2 has carried a heterozygous c.2401A>C mutation in KIT gene. The same mutation was not found in unaffected members. CONCLUSION: We have attained definite diagnosis for both families, which has facilitated genetic counseling and assisted reproduction for our patients and their family members.

[Mutation screening of the F VIII gene in 10 hemophilia A families].

OBJECTIVE: To identify the F VIII gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. METHODS: PCR, denaturinghigh performance liquid chromatogramphy (DHPLC) and DNA sequencing technologies were applied to screen the F VIII gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14(DXS 52), intron 13 (CA)n and EX18/Bcl I of the F VIII gene in the HA families. In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F VIII gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. RESULTS: Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism (SNP) were identified in 10 the HA families. Among them, c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT, c.4880_4881insA and c.5000G to A were novel mutations or polymorphism. No missense mutations c.878A G, c.1015A to G and c.6870G to T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined to in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. CONCLUSION: Six novel mutations, i.e., c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT and c.4880_4881insA, were identified in this study. PCR, DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.