Cloning and Functional Analysis of LtMYB305 in Liriodendron tulipifera

doi:10.13560/j.cnki.biotech.bull.1985.2023-1069

Abstract

Abstract:

【Objective】MYB305 as a member of the MYB R2R3 subfamily, plays an important role in plant nectary development, nectar protein expression, starch accumulation and hydrolysis, and flavonoid synthesis. Therefore, it is of great significance to study the expression profile and function of MYB305 for the molecular mechanism study of Liriodendron tulipifera floral nectary. 【Method】Having floral nectary of nectariferous plant L. tulipifera as materials, the anthocyanidin content in five stages of floral nectary was detected by spectrophotometry, LtMYB305 gene was cloned via RACE（rapid-amplification of cDNA ends）technology, and relative expressions among different tissues were analyzed by RT-qPCR assay. Then, subcellular localization of LtMYB305 protein was verified using tobacco leaves as materials, and the function of LtMYB305 gene was studied by genetic transformation approaches of wild type Arabidopsis thaliana（col）. 【Result】The anthocyanidin in the nectaries of L. tulipifera was accumulated from the late expanding stage, increased sharply at the early flowering stage, and reached the highest of 27.14 μg/g at the withering stage, which was consistent with the process of nectar secretion and coloring. The full-length of LtMYB305 was 931 bp encoding 198 amino acids and its protein was hydrophilic and non-transmembrane. LtMYB305 was expressed highly only in the nectary of L. tulipifera at anthesis stage, and hardly in other tissues. LtMYB305 protein was localized in the nucleus. After the overexpression of LtMYB305, the nectary groove of the lateral nectary disappeared and deepened, the expressions of AtMYB30, AtPIN, AtSWEET9 genes related to flower nectary were up-regulated, and the expressions of AtCRC, AtBOP1/2, AtMYB21, AtSWEET3/4/7 genes were down-regulated. 【Conclusion】The LtMYB305 has typical characteristics of transcription factor and is involved to the nectary development in L. tulipifera.

Key words: Liriodendron tulipifera, nectary, MYB305, gene cloning, functional analysis

LIU Huan-huan, YANG Li-chun, LI Huo-gen. Cloning and Functional Analysis of LtMYB305 in Liriodendron tulipifera[J]. Biotechnology Bulletin, 2024, 40(4): 179-188.

Figures/Tables 11

Fig. 1 Anthocyanidin content analysis during floral nectary growth of L. tulipifera * indicates significant difference at P<0.05 level; ** indicates significant difference at P<0.01 level. The same below

Table 1 Prediction of LtMYB305 protein subcellular localization in L. tulipifera by SignalP-4.0 Server %

蛋白名称 Protein name	可信度Reliability
蛋白名称 Protein name	叶绿体转运肽Chloroplast transit peptide, cTP	线粒体靶向肽Mitochondrial targeting peptide, mTP	分泌通路信号肽Secretory pathway signal peptide, SP	其他 Other
LtMYB305	9.40	4.90	8.00	94.70

Table 2 Prediction of LtMYB305 protein subcellular localization in L. tulipifera by WOLF PSORT

蛋白名称 Protein name	亚细胞结构Subcellular structure
蛋白名称 Protein name	细胞核 Nuclear	过氧化物酶体Peroxisome	叶绿体Chloroplast	液泡 Vacuole
MYB305	13	1	-	-

Fig. 2 Multiple alignment of the amino acid（A）sequences and phylogenetic tree analysis（B）of LtMYB305 in L. tulipifera with other MYB proteins

Fig. 3 Expression profiles of LtMYB305 genes in different tissues of L. tulipifera by semi-quantitative PCR（A）andRT-qPCR assays（B）

Fig. 4 Subcellular localization of LtMYB305 proteins in L. tulipifera

Fig. 5 Expression analysis of T1 generation of positive 35S::LtMYB305-overexpressed A. thaliana plants A: Semi-quantitative PCR analysis in eight lines of T1 generation positive plants; B: RT-qPCR analysis in eight lines of T1 generation positive plants; C: RT-qPCR analysis in three high-expressed lines of T1 generation positive plants; D: RT-qPCR analysis of eleven genes related to nectary development in three high-expressed lines of T1 generation positive plants

Fig. 6 Screening of positive 35S:: LtMYB305-overexpressed A. thaliana plants A: Detection of positive T2-T3 generation 35S::LtMYB305-overexpressed A. thaliana plants. B: Root observation of wild type and 35S::LtMYB305-overexpressed A. thaliana plants. C: Screening of seedling of wild type and 35S::LtMYB305-overexpressed A. thaliana plants

Fig. 7 Phenotype observation of 35S::LtMYB305-overexpressed A. thaliana A: Floral nectaries of wild type A. thaliana（col）plants. B: Floral nectaries 35S::LtMYB305-overexpressed A. thaliana plants. C: T1-T3 generation of 35S::LtMYB305 -overexpressed A. thaliana plants. LN: Lateral nectary

Fig. 8 Stem heights of 35S::LtMYB305-overexpressed A. thaliana plants

Fig. 9 SEM of floral nectary of 35S::LtMYB305-overexpressed A. thaliana plants A-D: Floral nectaries of wild type A. thaliana（col）plants. E-L: Floral nectaries of 35S::LtMYB305-overexpressed A. thaliana plants. MN: Medial nectary. LN: Lateral nectary. Sto: Stoma

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