北美鹅掌楸LtMYB305基因的克隆及功能分析

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

摘要/Abstract

摘要：

【目的】MYB305作为MYB家族R2R3亚族成员，对植物花蜜腺发育、花蜜蛋白表达、淀粉积累和水解、类黄酮合成发挥重要作用。因此，研究MYB305的表达模式及功能对于北美鹅掌楸花蜜腺调控分子机理研究具有重要意义。【方法】本文以蜜源植物北美鹅掌楸的花蜜腺为材料，通过分光光度计法测定5个时期花蜜腺的花青素含量，采用RACE技术克隆LtMYB305基因，利用RT-qPCR技术检测了该基因在北美鹅掌楸不同组织间相对表达量，以烟草叶片为材料验证LtMYB305蛋白定位，并以野生型拟南芥为材料进行遗传转化实验，研究LtMYB305基因功能。【结果】北美鹅掌楸花蜜腺的花青素从膨大后期开始积累，初放期含量急剧增加，败花期含量最高，达27.14 μg/g，与花蜜分泌、着色过程相一致。LtMYB305基因全长为931 bp，编码了198个氨基酸，其蛋白为亲水性蛋白和非跨膜蛋白；LtMYB305仅在北美鹅掌楸盛花期花蜜腺中高水平表达，在其他组织中几乎不表达。LtMYB305蛋白定位于细胞核。过表达LtMYB305基因的拟南芥出现侧蜜腺的“蜜腺沟”消失和加深表型，与花蜜腺相关基因AtMYB305、AtPIN6和AtSWEET9表达量上调，AtCRC、AtBOP1/2、AtMYB21、AtSWEET3/4/7基因表达量下调。【结论】LtMYB305具有典型转录因子特征，并且参与调控拟南芥花蜜腺的发育。

关键词: 北美鹅掌楸, 蜜腺, MYB305, 基因克隆, 功能分析

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

刘换换, 杨立春, 李火根. 北美鹅掌楸LtMYB305基因的克隆及功能分析[J]. 生物技术通报, 2024, 40(4): 179-188.

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.

图/表 11

图1 北美鹅掌楸不同时期花蜜腺的花青素含量分析 *表示在P<0.05水平上差异显著，**表示在P<0.01水平上差异显著。下同

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

表1 北美鹅掌楸LtMYB305蛋白亚细胞定位SignalP-4.0 Server预测分析

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

表2 北美鹅掌楸LtMYB305蛋白亚细胞定位WOLF PSORT预测分析

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	-	-

图2 北美鹅掌楸MYB305蛋白的氨基酸序列比对（A）和进化树构建（B）

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

图3 半定量PCR（A）和实时荧光定量PCR（B）检测北美鹅掌楸MYB305基因在不同组织中的表达模式

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

图4 北美鹅掌楸LtMYB305蛋白亚细胞定位

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

图5 超表达35S::LtMYB305拟南芥阳性植株T1代表达量分析 A：阳性植株T1代8个株系半定量PCR检测；B：阳性植株T1代8个株系实时荧光定量PCR检测；C：阳性植株T1代3个高表达株系实时荧光定量PCR检测；D：阳性植株T1代3个高表达株系的蜜腺发育相关基因实时荧光定量PCR检测

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

图6 过表达35S::LtMYB305拟南芥阳性植株筛选 A：过表达LtMYB305 T2-T3代阳性植株检测；B：野生型拟南芥和过表达植株的根系；C：野生型拟南芥和过表达植株幼苗筛选

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

图7 过表达35S::LtMYB305拟南芥表型观察 A：野生型拟南芥（col）花蜜腺；B：过表达35S::LtMYB305拟南芥花蜜腺；C：过表达LtMYB305拟南芥T1-T3代植株；LN：侧蜜腺

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

图8 过表达LtMYB305拟南芥阳性植株茎高

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

图9 过表达35S::LtMYB305拟南芥阳性植株的花蜜腺扫描电镜观察 A-D：野生型拟南芥（col）花蜜腺；E-L：过表达LtMYB305拟南芥花蜜腺；MN：主蜜腺；LN：侧蜜腺；Sto：气孔

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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