基于水稻的口服霍乱疫苗MucoRice-CTB 51A品系的下一代种子库与原始主种子库的全基因组和蛋白质组学比较分析。
Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine.
作者信息
Sasou Ai, Yuki Yoshikazu, Honma Ayaka, Sugiura Kotomi, Kashima Koji, Kozuka-Hata Hiroko, Nojima Masanori, Oyama Masaaki, Kurokawa Shiho, Maruyama Shinichi, Kuroda Masaharu, Tanoue Shinjiro, Takamatsu Narushi, Fujihashi Kohtaro, Goto Eiji, Kiyono Hiroshi
机构信息
Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Asahi Kogyosha Co., Ltd., Tokyo, Japan.
出版信息
BMC Genomics. 2021 Jan 19;22(1):59. doi: 10.1186/s12864-020-07355-7.
BACKGROUND
We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker-free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB.
RESULTS
We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy.
CONCLUSIONS
All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future.
背景
我们之前研发了一种基于水稻的霍乱腹泻口服疫苗，即黏液水稻-霍乱毒素B亚单位（MucoRice-CTB）。利用农杆菌介导的共转化，我们培育出了无选择标记的MucoRice-CTB品系51A，该品系在水稻基因组中插入了三个霍乱毒素B亚单位（CTB）基因拷贝和两个RNA干扰盒拷贝。我们确定了转基因在水稻第3和第12号染色体上的序列和位置。水稻中的主要过敏原蛋白α-淀粉酶/胰蛋白酶抑制剂在该品系中的表达低于野生型水稻。品系51A进行了五代自花授粉以固定转基因，由T5植株产生的第六代种子被定义为主种子库（MSB）。从部分MSB种子培育出T6植株，并进行自花授粉以产生T7种子（下一个种子库；NSB）。对NSB进行了检测，并将其全基因组和蛋白质组与MSB的进行了比较。
结果
我们对NSB和MSB的转基因进行了重测序，并确认了插入到第3和第12号染色体上的三个CTB基因的位置。NSB和MSB之间转基因的DNA序列相同。利用全基因组测序，我们比较了三个NSB样本和三个MSB样本的基因组序列，并通过聚类评估了单核苷酸多态性（SNP）和基因组结构变异的影响。在NSB和MSB样本之间未检测到功能上重要的突变（染色体上基因区域的SNP、易位、缺失或倒位）。通过鸟枪法串联质谱（shot-gun MS/MS）对NSB和MSB样本的盐溶性蛋白质进行分析，未检测到蛋白质丰度有显著差异。通过免疫荧光显微镜检查，未检测到NSB和MSB成熟种子中贮藏蛋白和CTB的表达模式有差异。
结论
所有分析均显示NSB和MSB样本之间没有显著差异。因此，在不久的将来，NSB可用于替代MSB。
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