GhHAM regulates GoPGF-dependent gland development and contributes to broad-spectrum pest resistance in cotton.

Cotton is a globally cultivated crop, producing 87% of the natural fiber used in the global textile industry. The pigment glands, unique to cotton and its relatives, serve as a defense structure against pests and pathogens. However, the molecular mechanism underlying gland formation and the specific role of pigment glands in cotton's pest defense are still not well understood. In this study, we cloned a gland-related transcription factor GhHAM and generated the GhHAM knockout mutant using CRISPR/Cas9. Phenotypic observations, transcriptome analysis, and promoter-binding experiments revealed that GhHAM binds to the promoter of GoPGF, regulating pigment gland formation in cotton's multiple organs via the GoPGF-GhJUB1 module. The knockout of GhHAM significantly reduced gossypol production and increased cotton's susceptibility to pests in the field. Feeding assays demonstrated that more than 80% of the cotton bollworm larvae preferred ghham over the wild type. Furthermore, the ghham mutants displayed shorter cell length and decreased gibberellins (GA) production in the stem. Exogenous application of GA3 restored stem cell elongation but not gland formation, thereby indicating that GhHAM controls gland morphogenesis independently of GA. Our study sheds light on the functional differentiation of HAM proteins among plant species, highlights the significant role of pigment glands in influencing pest feeding preference, and provides a theoretical basis for breeding pest-resistant cotton varieties to address the challenges posed by frequent outbreaks of pests.

[Study on optimizing extraction methods of Lamiophlomis rotata].

OBJECTIVE: To optimize different extraction methods of Lamiophlomis rotata Kudo. METHODS: With the content of total flavonoids as assay index, the effect of three extraction methods was compared and the optimal excracting technology was selected by orthogoral test. RESULTS: The contents of flavonoids and solids by ethanol extraction were more than that by the boiling water extraction and ultrasonic extraction. CONCLUSION: The ethanol extraction is the optimal extracting technology, which is to put 10g powder of Lamiophlomis into 200ml 60% alcohol, and then reflux extract for 25 min.

[Effects of slow release-fertilizers on yield, nutrient contents and quality of Coptis chinensis].

OBJECTIVE: To study the effects of four kind of slow-release fertilizers on yield and quality of Coptis chinensis. METHOD: One to three years C. chinensis was fertilized with slow-release fertilizers twice in April and in September. The yield and nutrient content along with quality of C. chinensis were measured after two years growth. RESULT AND CONCLUSION: All of the slow-release fertilizers increased the yield obviously, and the effect of SRF1 and SRF4 is the most significant. Comparing with control group, the N content in aerial part of 1-2 year-old C. chinensis treated with SRF1 and SRF4 was lower and P and K were higher than that of control group, and the N content in aerial part of 3 year-old C. chinensis treated with SRF1 and SRF4 was higher and P and K were higher than that of control group; The N content in the root of land 3 year-old C. chinensis treated with SRF1 and SRF4 showed no significant difference comparing with control group, and P and K were lower than that of control group, the N and P content in root of 2 year-old C. chinensis treated with SRF1 and SRF4 was higher and K were lower than that of control group. After two years growth berberine content of C. chinensis treated with SRF1, SRF2 and SRF3 were significantly increased, and total alkaloid content of C. chinensis treated with SRF1, SRF3 and SRF4 were significantly increased. We recommend that SRF4 is used as the special fertilizer for 1-year-old C. chinensis, and the SRF1 and SRF4 for 2-year-old C. chinensis, and the SRF1 for 3-year-old C. chinensis.

Construction of cDNA library of cotton mutant Xiangmian-18 library during gland forming stage.

Gossypol, a secondary metabolite stored in the glands of cotton, protecting cottonseed from consumption of human and monogastric animal. This ability is unique to the tribe Gossypieae. Although the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been investigated at molecular level. Here we described a simple and efficient method for constructing a normalized cDNA library from a cotton mutant, Xiangmian-18, during its pigments gland forming stage. It combined switching mechanism at 5'-end of RNA transcript (SMART) technique and duplex-specific nuclease (DSN) normalization methods. In a model experiment, double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into E. coli JM109 by electroporation. Counting the number of colonies, the titer of the original library was 5.86x10(5)cfu/ml in this library. Electrophoresis gel results indicated the fragments ranged from 800bp to 2kb, with the average size of 1400bp. Random picking clones showed that the recombination rate was 94%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of pigments gland cottons.

Molecular cloning and expression analysis of a RanBP2 zinc finger protein gene in upland cotton (Gossypium hirsutum L.).

Gossypol is an important resistant substance of Gossypium, and its storage organ is pigment gland. Although, the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been revealed up to now in molecular perspective. On the basis of differentially expressed cDNAs fragments at the stage of the cotton gland development using suppression subtractive hybridization (SSH), the complete cDNA sequence of a novel RanBP2 zinc finger protein (ZFP) gene was cloned by rapid amplification of cDNA ends (RACE) from upland cotton (Gossypium hirsutum L.), Xiangmian 18. The cotton RanBP2 ZFP cDNA (GenBank accession number: DQ173926) is 717 base pair (bp) long with an open reading frame encoding 139 amino acids, which encodes a 15.6 kDa protein. The cotton RanBP2 ZFP had three Ran-binding protein (RanBP) two zinc finger motifs and belonged to RanBP2 ZFP family. There is a 292-base non-coding sequence at 3' cDNA end, which includes polyA sequence. Sequence alignment analysis revealed that the cDNA nucleotide and its deduced amino acid sequence are moderately identical to the putative ZFP from other species. The mRNA expressing profiles of the novel ZFP gene was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The result showed that it expressed at different development stages of gland, including the undeveloped stage, developing stage, developed stage and cotyledon stage. However, with the development of pigment gland, the mRNA levels in the gland-developed seed and cotyledon were increased to about 1.5 and 2 folds of that in gland-undeveloped seed, respectively, which suggested that the novel ZFP played a role in the development of the cotton gland.