OsPLGG1 enhanced function of photorespiratory bypass
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摘要:
目的 在已构建GOC（乙醇酸氧化酶OsGLO、草酸氧化酶OsOXO和过氧化物酶OsCAT）光呼吸支路的工程水稻中，敲除乙醇酸转运体编码基因OsPLGG1a或OsPLGG1b，以优化光呼吸支路的改造，提高水稻光合效率。 方法 以GOC工程水稻为背景材料，通过CRISPR-Cas9技术分别敲除OsPLGG1a或OsPLGG1b基因，通过抗性筛选和测序鉴定osplgg1a-GOC和osplgg1b-GOC纯合敲除株系，并测定光合速率。 结果 获得敲除OsPLGG1a或OsPLGG1b的GOC代谢支路的纯合株系(osplgg1a-GOC和osplgg1b-GOC)。osplgg1a-GOC植株与ZH11背景中OsPLGG1a敲除突变体表型相似，均表现出黄化矮小生长抑制的表型；osplgg1b-GOC植株相比GOC水稻的净光合速率提高，说明OsPLGG1b突变有利于叶绿体中滞留乙醇酸，增加GOC支路的代谢通量，进一步提高叶绿体中的CO2浓度。 结论 相对于GOC水稻，osplgg1b-GOC部分株系的净光合速率和气孔导度增加，暗示敲除OsPLGG1b可用于光呼吸代谢支路的优化。 -
关键词:
- OsPLGG1 /
- 光呼吸 /
- GOC代谢支路 /
- 乙醇酸/甘油酸转运体 /
- 水稻
Abstract:Objective The deletion of either OsPLGG1a or OsPLGG1b in the GOC rice engineering optimizes the photorespiration pathway, thereby enhancing rice photosynthesis efficiency and optimizing photorespiration metabolic engineering. Method The knockout vectors osplgg1a-Cas9 or osplgg1b-Cas9 were constructed and transformed into GOC rice, respectively, to obtain GOC transgenic homozygous lines with deletion of OsPLGG1a or OsPLGG1b, and the photosynthesis rate was measured. Result Transgenic homozygous lines with the GOC rice of OsPLGG1a or OsPLGG1b knocked out were obtained. The phenotypes of osplgg1a-GOC plants were similar to osplgg1a, both of which showed stunted growth. Compared with GOC rice, the net photosynthetic rate of osplgg1b-GOC plants increased, indicating that OsPLGG1b mutation was conducive to the retention of chloroplast glycolate, increased the metabolic flux through the GOC bypass, reduced the photorespiration metabolism of plants, and further increased the CO2 concentration in chloroplasts. Conclusion Compared with GOC rice, the net photosynthetic rate and stomatal conductance of osplgg1b-GOC plants were increased, suggesting that the elimination of OsPLGG1b could be used to optimize the photorespiratory metabolic pathway. -
Key words:
- OsPLGG1 /
- photorespiration /
- GOC metabolic pathway /
- glycolate/glycerate transporter /
- rice
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图 1 osplgg1-Cas9-GOC的编辑位点
核苷酸划线“（delete-Xbp）”代表删除X核苷酸；“……”表示与野生型相同的部分序列。靶点设置在“CCN”或“NGG”（PAM）。黑色数字表示编码区中ATG的核苷酸位置。
Figure 1. Edit site analysis of osplgg1-Cas9-GOC
Nucleotide underscore "(delete-x bp)" stands for deleting X nucleotides; "......" represents the same partial wild-type sequence. The target was set at "CCN or NGG" (PAM). The black numbers indicate the nucleotide positions of ATG in ORF.
图 2 osplgg1a-GOC和osplgg1b-GOC植株的幼苗期表型
A、C：osplgg1a-GOC和osplgg1b-GOC表型；B、D：osplgg1a-GOC和osplgg1b-GOC株高；E：叶片灌浆期表型；F：叶绿素含量。A和C为osplgg1a-GOC和osplgg1b-GOC在空气环境条件下培养5周后的表型，在含有木村B营养液的培养箱内生长。根据Duncan法进行样本间差异显著性分析，不同小写字母表示不同植株之间差异显著（P ＜ 0.05）。图3、4同。
Figure 2. Seedling phenotype of osplgg1a-GOC and osplgg1b-GOC mutations
A, C: phenotype of osplgg1a-GOC and osplgg1b-GOC, plants grown under ambient for 5 weeks; B, D: plant height of osplgg1a-GOC and osplgg1b-GOC; E: phenotype; F: chlorophyll content. Plants were grown in the growth chamber in Kimura B nutrient solution. Means denoted by the same letter did not significantly differ at P ＜ 0.05 according to Duncan’s multiple range tests. Same for Fig.3, 4.
图 3 osplgg1b-GOC转基因植株的表型分析
A：osplgg1b-GOC转基因植株在灌浆期植株表型观察与完熟期穗形态观察；B：株高、分蘖数、剑叶长形态指标分析；C：结实率；D：穗粒数； E：穗长，n=30。
Figure 3. Phenotypic analysis of osplgg1b-GOC transgenic plants
A: phenotypic observation and panicle morphology observation of osplgg1b-GOC transgenic plants at grain filling stage; B: morphological index analysis of plant height, tiller number and length of flag leaf; C: seed setting rate; D: grain number of per panicle; E: panicle length, n=30.
图 4 osplgg1b-GOC转基因植株的光合指标分析
Figure 4. Analysis of photosynthesis indicators in osplgg1b-GOC plants
表 1 引物信息
Table 1. Primers used in this study
引物名称
Primer name引物序列（5′-3′）
Primer sequence （5′-3′）目的
PurposeCas9-F GAACGGTCGTAAGAGGATGC 无标记检测
Unmarked detectionCas9-R GGTGATGGACTGGTGGATGAG OsPLGG1a-pYLsgRNA-OsU6a-F1 CGTAGCGAGGCAATCATGCGTTTTAGAGCTAGAAAT 载体构建
Carrier constructionOsPLGG1a-pYLsgRNA-OsU6a-R1 GCATGATTGCCTCGCTACGCGGCAGCCAAGCCAGCA OsPLGG1a-pYLsgRNA-OsU6b-F2 TGCGACAAAGACAGCACCCCGTTTTAGAGCTAGAAAT OsPLGG1a-pYLsgRNA-OsU6b-R2 GGGGTGCTGTCTTTGTCGCACAACACAAGCGGCAGC OsPLGG1b-pYLsgRNA-OsU6a-F1 AGCCATGGGGACGGAATGCTGTTTTAGAGCTAGAAAT OsPLGG1b-pYLsgRNA-OsU6a-R1 AGCATTCCGTCCCCATGGCTCGGCAGCCAAGCCAGCA OsPLGG1b-pYLsgRNA-OsU6b-F2 CCCAGGCCCAGAATTCAAGGTTTTAGAGCTAGAAAT OsPLGG1b-pYLsgRNA-OsU6b-R2 CTTGAATTCTGGGCCTGGGCAACACAACGGCAGC M1：OsPLGG1a-Cas9-85F TCATCCACTGTCACTGCCACTG CRISPR/Cas9 突变位点检测
Detection of CRISPR/Cas9
mutation sitesM2：OsPLGG1a-Cas9-2187R GCACTACCTTTCTCTGCTTGA M3：OsPLGG1b-Cas9-2F TGGAACAGTGACGGCAGTTG M4：OsPLGG1b-Cas9-1795R GAGGTAATCACCTGGACAACC 
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