PtoMYB61对毛白杨木质素合成及耐盐性的影响

doi:10.13560/j.cnki.biotech.bull.1985.2025-0006

摘要/Abstract

摘要：

目的研究PtoMYB61对毛白杨（Populus tomentosa）次生细胞壁形成和胁迫防御的影响，为木材发育和抗逆性育种奠定基础。方法使用RT-PCR法从毛白杨中克隆PtoMYB61基因。进化树分析及同源性比对预测其生物学功能。利用RT-qPCR分析PtoMYB61的组织表达特异性及对生物和非生物胁迫的响应。叶盘法转化毛白杨获得PtoMYB61过表达及敲除株系。采用组织切片、甲苯胺蓝染色、木质素含量测定及次生细胞壁生物合成途径中关键酶基因的表达量检测来分析PtoMYB61对杨树次生发育的影响。利用150 mmol/L NaCl处理转基因株系，通过表型观察及生理指标测定分析PtoMYB61对毛白杨耐盐性的影响。结果 PtoMYB61基因编码了一个由309个氨基酸组成的R2R3-MYB转录因子，其在腋芽、叶和茎中有较高表达，且受到盐、ABA和真菌病的诱导。与野生型相比，过表达和敲除株系的生长表型没有明显差异，但PtoMYB61过表达植株的木质部细胞层数增多，木质素含量显著增加，次生细胞壁生物合成关键酶基因表达水平上调，而敲除PtoMYB61导致木质部细胞层数减少，木质素含量降低，及次生细胞壁生物合成关键酶基因下调。盐处理发现，与野生型相比，PtoMYB61敲除植株对盐胁迫更为敏感，而过表达株系则具有一定的耐受性。结论 PtoMYB61响应盐胁迫的诱导，并通过调控杨树次生细胞壁发育进而影响杨树对盐胁迫的耐受性。

关键词: 杨树, 转录因子, PtoMYB61, 次生细胞壁, 耐盐性

Abstract:

Objective The effects of PtoMYB61 on secondary cell wall formation and stress defense of Populus tomentosa were studied, so as to lay a foundation for studying wood development and stress resistance breeding. Method RT-PCR was used to clone the PtoMYB61 gene from P. tomentosa. Phylogenetic tree analysis and homology comparison was to predict its biological function. RT-qPCR was applied to analyze the tissue expression specificity of PtoMYB61 and its response to biotic and abiotic stresses. Leaf disc method was to transform P. tomentosa and the lines of overexpressed and knocked-out PtoMYB61 were obtained transformed. Tissue section, toluidine blue staining, lignin content determination, and expressions of key enzyme genes in secondary cell wall biosynthesis pathway were used to analyze the effect of PtoMYB61 on poplar secondary development. Transgenic lines were treated with 150 mmol/L NaCl, and the effects of PtoMYB61 on the tolerance of P. tomentosa to salt were analyzed by phenotypic observation and physiological index determination. Result PtoMYB61 gene encodes a R2R3-MYB transcription factor consisting of 309 amino acids, which is highly expressed in axillary buds, leaves and stems, and is induced by salt, ABA and fungal diseases. Overexpressed and knocked-out lines have no significant difference in growth phenotype compared with wild type, but the number of xylem cells in the overexpressed plants of PtoMYB61 increase, the lignin content significantly increases, and the expressions of key enzymes in secondary cell wall biosynthesis are up-regulated, while knocking PtoMYB61 out leads to the decrease of xylem cell layers, the decrease of lignin content and the down-regulation of key enzymes in secondary cell wall biosynthesis. Salt treatment shows that compared with wild type, PtoMYB61-knockout plants are more sensitive to salt stress, while the overexpressed plants have certain tolerance. Conclusion PtoMYB61 responds to the induction of salt stress and influences the tolerance of poplar to salt stress by regulating the development of secondary cell wall.

Key words: poplar, transcription factor, PtoMYB61, secondary cell wall, salt tolerance

李小欢, 陈相宇, 陶麒宇, 朱玲, 唐铭, 姚银安, 汪丽君. PtoMYB61对毛白杨木质素合成及耐盐性的影响[J]. 生物技术通报, 2025, 41(6): 284-296.

LI Xiao-huan, CHEN Xiang-yu, TAO Qi-yu, ZHU Ling, TANG Ming, YAO Yin-an, WANG Li-jun. Effects of PtoMYB61 on Lignin Biosynthesis and Salt Tolerance in Populus tomentosa[J]. Biotechnology Bulletin, 2025, 41(6): 284-296.

图/表 8

图1 PtoMYB61的系统发育分析及氨基酸序列比对A：PtoMYB61的蛋白进化树分析；B：PtoMYB61氨基酸同源性分析

Fig. 1 Phylogenetic analysis and amino acid sequence alignment of PtoMYB61A: Protein phylogenetic tree analysis of PtoMYB61; B: amino acid homology analysis of PtoMYB61

图2 PtoMYB61表达模式的特异性分析A：荧光定量分析PtoMYB61在不同组织中的表达量，R：根，S：茎，YL：幼叶，ML：成熟叶，P：叶柄，AB：顶芽，LB：腋芽；星号表示使用 Student t 检验的显著差异（*P<0.05；**P<0.01），n=3，下同

Fig. 2 Specificity analysis of PtoMYB61 expression patternA: Quantitative analysis of the expression of PtoMYB61 in different tissues by fluorescence, R: root, S: stem, YL: young leaf, ML: mature leaf, P: petiole, AB: terminal bud, LB: axillary bud. Asterisks indicate significant differences using Student’s t-test ( *P<0.05; **P<0.01 ), n=3, the same below

图3 PtoMYB61转基因植株的阳性鉴定A：35S：：PtoMYB61过表达株系的阳性鉴定电泳图，不同的数字代表独立的转基因株系；B：RT-qPCR分析35S：：PtoMYB61过表达株系中PtoMYB61的相对表达量；C： CRISPR/Cas9定点编辑PtoMYB61基因的靶点示意图，T1-T3代表3个靶点的设计区域；D：PtoMYB61突变株系的测序分析，图片显示3、4、5三个独立株系中间的碱基缺失情况

Fig. 3 Positive identification of transgenic plants of PtoMYB61A: Positive identification electropherogram of 35S::PtoMYB61 overexpressed plants, different numbers represent independent transgenic lines; B: RT-qPCR quantitative results of overexpressed strains; C: schematic diagram of CRISPR/Cas9 targeted editing of PtoMYB61, T1 to T3 represent the design areas of three targets; D: Sequencing analysis of PtoMYB61 mutant lines, the picture shows the base deletion in the middle of 3, 4 and 5 independent lines

图4 PtoMYB61转基因株系的形态学观察A：生长9周的毛白杨植株，标尺=10 cm；B：生长9周的毛白杨植株根，标尺=10 cm；C：4-9周的毛白杨植株高度统计；D：4-9周的毛白杨植株离地5 cm茎直径统计；E：生长11周的毛白杨根长；F：生长11周的毛白杨根部表面积

Fig. 4 Morphological observation of transgenic lines of PtoMYB61A: 9-week-old Populus tomentosa, scale bar=10 cm; B: roots of 9-week-old P. tomentosa plant, scale bar=5 cm; C: height statistics of P. tomentosa from 4 weeks to 9 weeks; D: statistics of stem diameter of P. tomentosa 5 cm above the ground from 4 weeks to 9 weeks; E: the root length of P. tomentosa growing for 11 weeks; F: root surface area of P. tomentosa growing for 11 weeks

图5 PtoMYB61转基因株系杨树木质素及总黄酮含量的检测A：WT植株和PtoMYB61转基因株系在体式显微镜下第5节间和第7节间的茎横切面；B：第4/5/7茎节的木质部细胞层数统计，n=10；C：WT和转基因植株茎中木质素含量，n=3；D：WT和转基因植株叶中的总黄酮含量，误差线表示均值±SD，不同的小写字母表示基于单因素方差分析后进行Tukey’s post hoc test的显著差异（P<0.05），下同

Fig. 5 Detection of lignin and total flavonoids in transgenic lines of PtoMYB61A: Cross-section of the stem of the 5th and 7th internode of WT plant and PtoMYB61 transgenic line under the stereomicroscope; B: statistics of xylem cell layers in the 4th, 5th and 7th stem nodes, n=10; C: lignin content in stems of WT and transgenic plants, n=3; D: total flavonoids content in leaves of WT and transgenic plants. The error line indicates the mean SD, and different lowercase letters indicate the significant difference of Tukey’s post hoc test based on one-way ANOVA (P<0.05), the same below

图6 转基因杨树茎中次生壁生物合成基因的表达分析A：木质素生物合成基因；B：纤维素生物合成基因；C：木聚糖合酶基因

Fig. 6 Expression analysis of secondary cell wall biosynthesis genes in transgenic poplar stemsA: Lignin biosynthesis genes; B: cellulose biosynthesis genes; C: xylan synthase gene

图7 盐处理对PtoMYB61过表达及敲除株系的影响A：150 mmol/L NaCl 处理7 d后的表型图，标尺=10 cm；B：150 mmol/L NaCl处理4 d后，DAB 染色结果图，标尺=5 cm

Fig. 7 Phenotype of PtoMYB61 over-expressing and knocking-out lines under salt stressA: Phenotypic map after 7 d of 150 mmol/L NaCl treatment, bar=10 cm; B: DAB staining results after four days of 150 mmol/L NaCl treatment, bar=5 cm

图8 PtoMYB61在盐胁迫前后的生理指标

Fig. 8 Physiological indexes of PtoMYB61 before and after salt stress

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