Evaluation of salt tolerant performance of BpCHS3 transgenic plants in Betula platyphylla
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摘要:
目的查耳酮合成酶（CHS）是黄酮类化合物生物合成途径中的关键酶，其高表达可以促进黄酮类化合物积累，增强植物抵抗盐碱、干旱等非生物胁迫的能力，开展转BpCHS3白桦耐盐性分析，为阐明该基因的功能提供参考。 方法以前期获得的转BpCHS3白桦为试材，进行转基因白桦qRT-PCR及Northern Blot检测，同时测定了叶片中花青素质量分数。NaCl胁迫处理下，分别测定转BpCHS3白桦的盐害指数、叶绿素荧光参数及光合参数。采用qRT-PCR技术，测定转基因株系的类黄酮代谢途径中CHS下游5个关键酶基因以及BpCHS家族成员相对表达量。 结果qRT-PCR及Northern Blot检测显示，导入的目标基因BpCHS3在mRNA水平能够表达。转基因白桦组培生根苗在0.3%NaCl胁迫第25天，对照（WT）株系盐害指数高达83%，而转基因白桦平均盐害指数仅为39%；白桦转基因盆栽苗在0.4%NaCl胁迫后，叶绿素荧光参数及光合参数测定显示，NaCl胁迫第9天多数转基因株系最大光化学效率（Fv/Fm）仍为正常值，而WT株系降至0.66，胁迫第9天时实际光化学效率（ΦPSII）和光化学猝灭系数（qP）呈下降趋势，但WT株系的降幅高于转基因株系；NaCl胁迫6 d时，5个转基因株系的净光合速率（Pn）平均值仍高于WT的43.47%。认为盐胁迫下BpCHS3基因在白桦中的过量表达能够维持其较高的光电子传递活性，提高PSⅡ反应中心原初光能转换效率，同时也能维持较高的净光合效率。分别以转BpCHS3白桦cDNA为模板qRT-PCR分析显示，相对WT株系CHS下游的5个关键酶基因在转基因株系中均呈不同程度的上调表达，BpCHS家族成员中只有BpCHS3表达量显著上调，而 BpCHS1和BpCHS2均呈下调表达或与WT株系差异不显著。BpCHS3过表达株系花青素质量分数分析发现，转基因白桦叶片中花青素质量分数均显著低于WT株系，推测BpCHS3的过量表达，对另外2个CHS家族成员产生共抑制，且影响花青素的合成。 结论转BpCHS3白桦的耐盐性提高，与花青素质量分数多少无关，BpCHS3的过表达可能促进其他黄酮类化合物的积累，从而增强白桦的耐盐能力。 Abstract:ObjectiveChalcone synthase (CHS) is a key enzyme in the biosynthesis pathway of flavonoids. Overexpression of CHS promotes an accumulation of flavonoids and enhances the ability of plants to resist abiotic stresses such as salinity and drought. The objective of the study is to elucidate the function of BpCHS3 to salt tolerance. MethodOur previously obtained transgenic birch plants of BpCHS3 were used in this study. The content of anthocyanins in leaves, salt stress index, chlorophyll fluorescence and photosynthetic parameters under NaCl stress were determined. qRT-PCR and Northern Blot were used to determine the relative expression levels of five key enzyme genes of downstream of CHS and BpCHS family members in the flavonoid metabolic pathway. ResultThe wild type (WT) had a salt damage index of 83% in the 25th day under 0.3% NaCl stress, whereas transgenic birch was only 39%. The chlorophyll fluorescence and photosynthetic parameters of transgenic Betula platyphylla seedlings under 0.4% NaCl stress showed that the maximum light energy conversion efficiency (Fv/Fm) for most transgenic lines was still normal in the 9th day, while Fv/Fm was reduced to 0.66 and the actual photochemical efficiency (ΦPSII) and photochemical quenching coefficient (qP) showed a decreasing trend for WT. The WT strain had a higher decline than the transgenic lines. The mean value of net photosynthetic rate (Pn) for five transgenic lines was still higher than 43.47% of WT under salt stress in 6 days. It is believed that the overexpression of BpCHS3 gene in Betula platyphylla under salt stress can maintain its higher photoelectron transport activity, increase the primary light energy conversion efficiency of PSII reaction center, and maintain a higher net photosynthetic efficiency. qRT-PCR and Northern Blot showed that the introduced target gene BpCHS3 was expressed in BpCHS3 transgenic lines. All of them showed up-regulated expression in different levels. Only BpCHS3 expression was significantly up-regulated in BpCHS family members. Both BpCHS1 and BpCHS2 were down-regulated or not significantly different from WT lines. Anthocyanin content of BpCHS3 overexpression lines was significantly lower than that of WT lines. It is speculated that overexpression of BpCHS3 results in co-suppression of two other CHS family members affects the synthesis of anthocyanins. ConclusionThe salt tolerance of BpCHS3 transgenic birch was increased, which was not related to the content of anthocyanin. The overexpression of BpCHS3 may promote the accumulation of other flavonoids, thus enhancing the salt tolerance of Betula platyphylla. -
Key words:
- Betula platyphylla /
- BpCHS3 /
- genetic transformation /
- salt tolerance
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图 1 转BpCHS3白桦qRT-PCR检测
Figure 1. Relative quantification of each transgenic line BpCHS3 gene for Betula platyphylla
图 2 转BpCHS3基因白桦Northern Blot检测图谱
1. WT；2 ~ 4. CHS3-1、CHS3-2、CHS3-4转基因株系。1, WT; 2−4, CHS3-1, CHS3-2, CHS3-4 transgenic lines.
Figure 2. Northern Blot pattern of 35S :: BpCHS3 from transgenic birch
图 3 盐胁迫下参试株系荧光参数及Pn比较
Fv/Fm. 最大光能转化效率Maximum light energy conversion efficiency；ΦPSII. 实际光化学效率Actual photochemical efficiency；qP. 光化学猝灭系数Photochemical quenching coefficient；Pn. 净光合速率Net photosynthetic rate
Figure 3. Comparison of fluorescence parameters and Pn of the tested birch under salt stress
图 4 转基因株系与WT株系叶片花青素质量分数比较
Figure 4. Comparison of anthocyanin mass fractions in leaves between transgenic lines and WT lines
图 5 黄酮类代谢途径关键酶基因qRT-PCR分析
Figure 5. qRT-PCR analysis of key enzyme genes in flavonoid metabolic pathway
表 1 荧光定量PCR引物序列
Table 1. Primer sequences for fluorescent quantitative-PCR
引物名称 Primer name 上游引物序列 Forward primer sequence (5′−3′) 下游引物序列 Reverse primer sequence (5′−3′) 18SrRNA ATCTTGGGTTGGGCAGATCG CATTACTCCGATCCCGAAGG BpCHS1 CCGTGGAGGAAATCCGAAAGGCTC CGCTCTTGGTGATCCGGAAGTAATA BpCHS2 ATGGCGTCCGTCGAAGAAATATTTAA TCGTCTTGGTAGATATAGTTGGATGG BpCHS3 GGCATCTCGGACTGGAACTC CACAACGGTCTCGACGGTAA BpF3′H CTTGAACCACCGCAACCTCAC CCTCGTCCTCGACCCGAATT BpCHI GTATTTGGAGGATACCGCCGTGC GCAAGATCATTGTCACCCGTGTG BpDFR1 TGCCGCCTAGTCTTATTACAGCAC GTGAATTGTGGCATCGGCGG BpDFR2 CATTGGCGTCTTCCACGTGGCTAC CGACAGCTGATCCACTGGTAGTGT BpANR1 AAGGATCGACAATTCCACAAGCAGG CGTATAGCTTGTTGGTGCTGGAGAA BpANR2 GGGGGAAGATTGTGATACGGAGGAT GCGAAATCGTAGAGCTTGCTGGTGC BpANR3 CGACGAGCAGGCAAGTGACTTTCTC TCGTAGAGCCTGCCGGTGCTGGAG BpANS TACTACCCGATCTGCCCTCAGC GGAACACATTTCGCCGTCACCC 
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表 2 NaCl胁迫下转基因白桦生根情况
Table 2. Rooting of transgenic birch under salt stress
株系
Strain平均主根数
Average number of primary roots平均主根长
Average main root length/cm生根率
Rooting rate/%盐害指数
Salt damage index/%WT 1.2 0.27 ± 0.02c 40 83 CHS3-1 3.2 1.76 ± 0.03a 100 43 CHS3-2 2.8 1.66 ± 0.02b 100 35 注：平均主根长表示方式为“平均值 ± 标准差”，同列不同字母表示差异显著（P < 0.05）。 Notes: data of average main root length were mean ± SD，different lowercases in the same column represent significant differences (P < 0.05).
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