CclSAUR49对柑橘生长及类柠檬苦素代谢的影响

doi:10.16420/j.issn.0513-353x.2023-0117

摘要/Abstract

摘要：

SAUR（Small auxin up-regulated RNA）为植物特有的早期生长素响应基因。为了研究柑橘中CclSAUR49参与调节其类柠檬苦素代谢的功能，用湖北早实枳（Poncirus trifoliata Raf.）的上胚轴进行遗传转化，获得6株CclSAUR49的过量表达植株（OE1 ~ OE6）和2株CclSAUR49的干扰表达植株（Ri1和Ri2），观察比较转基因和野生型植株的生长表现，利用电镜扫描和半薄切片分别观察叶片的表皮显微结构和组织结构，并检测了叶片中的生长素和类柠檬苦素含量。6个过量表达植株的形态表现存在差异，其中OE2与野生型相比枝梢增长了23.21%，节间数量增多，叶片长度增加了17.97%；2个干扰表达植株（Ri1和Ri2）枝梢生长量减少23.95%和46.82%；2/3过量表达植株的叶片主叶脉直径增大，而Ri2的减小。6株过量表达植株叶片中CclSAUR49的表达水平显著上调，是野生型的2.47 ~ 73.29倍，2株干扰表达植株叶片中CclSAUR49的表达都受到明显抑制，表达水平为野生型的22.66%和47.22%。转基因植株叶片中CclSAUR49的表达水平与其生长素含量呈极显著正相关，与生长素生物合成（YUC6和YUC10）、转运（PIN3）和信号转导（CiARF7和CiARF9）基因的表达水平呈极显著正相关。4个过量表达植株叶片中类柠檬苦素含量降低，1个干扰表达植株（Ri1）叶片中类柠檬苦素含量显著提高。在干扰表达植株中，类柠檬苦素的含量与生长素的含量呈显著负相关。以上结果表明，CclSAUR49能促进柑橘植株的生长发育，通过影响生长素的生物合成、转运和信号转导途径上基因的表达调控植物组织中的生长素水平，从而影响类柠檬苦素的代谢。

关键词: 柑橘, CclSAUR49, 遗传转化, 生长素, 类柠檬苦素

Abstract:

SAUR（Small auxin up-regulated RNA） is a plant-specific early auxin response gene. In order to study the function of CclSAUR49 in regulating limonoid metabolism in citrus，six CclSAUR49 overexpression plants（OE1-OE6）and two RNA interference plants（Ri1 and Ri2）were generated through genetic transformation with epicotyl of Poncirus trifoliata Raf. The growth performance of transgenic and wild type plants was observed and compared. The epidermal microstructure and tissue structure of leaves were observed by electron microscopy and semi-thin sections，respectively. The contents of auxin and limonoids in the leaves were also detected. The morphological performance of the six overexpression plants was different. Compared with the wild type，the shoot length of OE2 increased by 23.21%，the number of internodes increased，and the leaf length increased by 17.97%. The branch growth of two RNA interference expression plants（Ri1 and Ri2）was less than 23.95% and 46.82%. The main leaf vein diameter of 2/3 overexpression plants increased，while Ri2 decreased. The expressions level of CclSAUR49 in the leaves of six overexpression plants were significantly up-regulated，which was 2.47-73.29 times of the wild type. The expression of CclSAUR49 in the leaves of two RNA interference expression plants were significantly inhibited，and the expression levels were 22.66% and 47.22% of the wild type. The expression level of CclSAUR49 in the leaves of transgenic plants was significantly positively correlated with its auxin content，and was significantly positively correlated with the expression levels of auxin biosynthesis（YUC6 and YUC10），transport（PIN3）and signal transduction（CiARF7 and CiARF9）genes in the transgenic plants. The content of limonoids in the leaves of four overexpression plants decreased，and the content of limonoids in the leaves of one RNA interference expression plant（Ri1）increased significantly. In the RNA interference expression plants，the content of limonoids was significantly negatively correlated with the content of auxin. The above results indicate that CclSAUR49 can promote the growth and development of citrus plants，and regulate the auxin level in plant tissues by affecting the expression of genes in auxin biosynthesis，transport and signal transduction pathways，thereby affecting the metabolism of limonoids.

Key words: citrus, CclSAUR49, genetic transformation, auxin, limonoids

易倩, 张曼曼, 汪小利, 冯继鹏, 朱世平, 王福生, 赵晓春. CclSAUR49对柑橘生长及类柠檬苦素代谢的影响[J]. 园艺学报, 2024, 51(3): 479-494.

YI Qian, ZHANG Manman, WANG Xiaoli, FENG Jipeng, ZHU Shiping, WANG Fusheng, ZHAO Xiaochun. CclSAUR49 Affects Growth and Limonoids Metabolism in Citrus[J]. Acta Horticulturae Sinica, 2024, 51(3): 479-494.

图/表 15

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引物名称 Primer name	正向引物序列（5′-3′） Forward primer sequence	反向引物序列（5′-3′） Reverse primer sequence
SAU49OE	ATTTAAATATGGCTATTCATGTACCTGGAATC	GGATCCATATGATTCATTCAAGCTGGAAGT
M13Rf	TTCACACAGGAAACAGCTATGAC
SAUR49-RNAi	TCTAGAATTTAAATATGGCTATTCATGTACCTGGAATC	GGATCCATTTAAATTCATGATTCATTCAAGCTGGAA
59MSac-F	CACGCTCGAGTATAAGAGCTCT
59MBcu1-R		GGACTAGTCCCGGGTCTTAAT
SAUR49	ACATCCACAGTTGTGCCAAAAG	CAAACCCGAATTCTTCCTCAGC
YUC1	CACAATGCAATCCCTCACATG	ACCTTAACGGAAACCACCTGA
YUC2	CGCCTTGTGGATCAATTTTTA	AATGTTGCCGCTTCTGATCTT
YUC3	TGTGTTGGACATTGGTGCTTT	ACATTGCTTCGGTACCCAGTT
YUC5	GATTACAAGTCCGGCGAAAA	TGATGGAGAGGCATTGTGGTT
YUC6	AGAGGCAAAAGGGTTTTGGTT	CGAAAGTTGATTTCCCCAGC
YUC10	GGCTTTTGAATGGGATGATGA	ATTTGATTTGATGGCCCAACT
PIN1	GGAGCTGGAGCTAGGGATCTT	CTCCTTTTCCTCCTTGTGGTG
PIN2	GAGTGGCGAGCTTTTTAACG	ACCACCACCAACACTCTCCTT
PIN3	AAAACGGGGATTTTGGTAGG	GCGGGTCCACCTTCTTTATGA
PIN5	GATGAAGCTTGTGTGGCTGAA	AATGCCACCTGTTTGACACAA
PIN6	GAAATGGACGCGGTTACTGTT	GCCAGCTTGCCTATCTCCTTG
CiARF1	CTTGTAACCCTTCCTCGTG	GAAGAACGGAGAACCCAC
CiARF2	GTCAACTAATCAGGTCGCA	GGAAGACACTCATCAGCAT
CiARF7	AGTTGGAATAGAGCGTCG	GGAAGATTTGGATAGTTTGG
CiARF9	GGATTCCGTTGTTTCGTC	AATTCCTGAGTTGGGGTT
CiARF10	TGAAGAATGAGACTCGCTA	AACCTGTTCGCTATGACC
CiARF12	ACAGCCAAGATGATAAAGC	AATTCCCAACTTCCAGACT
CiARF13	TCAGAGGATGTAGGTCGCA	CAATCTTCTAGCCGTTTTCA
CiARF18	GGTTCTAAATGGCGGTCGT	CGACGCAGGAATGTCAATG
CiOSC	CTTTTGGCCTGGTGACTATGGT	CATTTCCCTTCGATGTTCCACT
CiMYB42	GGGCGAACCGATAACGAGAT	GCTGGAGTCTGGCAGCAAAT
GAPDH	GGAAGGTCAAGATCGGAATCAA	CGTCCCTCTGCAAGATGACTCT

引物名称 Primer name	正向引物序列（5′-3′） Forward primer sequence	反向引物序列（5′-3′） Reverse primer sequence
SAU49OE	ATTTAAATATGGCTATTCATGTACCTGGAATC	GGATCCATATGATTCATTCAAGCTGGAAGT
M13Rf	TTCACACAGGAAACAGCTATGAC
SAUR49-RNAi	TCTAGAATTTAAATATGGCTATTCATGTACCTGGAATC	GGATCCATTTAAATTCATGATTCATTCAAGCTGGAA
59MSac-F	CACGCTCGAGTATAAGAGCTCT
59MBcu1-R		GGACTAGTCCCGGGTCTTAAT
SAUR49	ACATCCACAGTTGTGCCAAAAG	CAAACCCGAATTCTTCCTCAGC
YUC1	CACAATGCAATCCCTCACATG	ACCTTAACGGAAACCACCTGA
YUC2	CGCCTTGTGGATCAATTTTTA	AATGTTGCCGCTTCTGATCTT
YUC3	TGTGTTGGACATTGGTGCTTT	ACATTGCTTCGGTACCCAGTT
YUC5	GATTACAAGTCCGGCGAAAA	TGATGGAGAGGCATTGTGGTT
YUC6	AGAGGCAAAAGGGTTTTGGTT	CGAAAGTTGATTTCCCCAGC
YUC10	GGCTTTTGAATGGGATGATGA	ATTTGATTTGATGGCCCAACT
PIN1	GGAGCTGGAGCTAGGGATCTT	CTCCTTTTCCTCCTTGTGGTG
PIN2	GAGTGGCGAGCTTTTTAACG	ACCACCACCAACACTCTCCTT
PIN3	AAAACGGGGATTTTGGTAGG	GCGGGTCCACCTTCTTTATGA
PIN5	GATGAAGCTTGTGTGGCTGAA	AATGCCACCTGTTTGACACAA
PIN6	GAAATGGACGCGGTTACTGTT	GCCAGCTTGCCTATCTCCTTG
CiARF1	CTTGTAACCCTTCCTCGTG	GAAGAACGGAGAACCCAC
CiARF2	GTCAACTAATCAGGTCGCA	GGAAGACACTCATCAGCAT
CiARF7	AGTTGGAATAGAGCGTCG	GGAAGATTTGGATAGTTTGG
CiARF9	GGATTCCGTTGTTTCGTC	AATTCCTGAGTTGGGGTT
CiARF10	TGAAGAATGAGACTCGCTA	AACCTGTTCGCTATGACC
CiARF12	ACAGCCAAGATGATAAAGC	AATTCCCAACTTCCAGACT
CiARF13	TCAGAGGATGTAGGTCGCA	CAATCTTCTAGCCGTTTTCA
CiARF18	GGTTCTAAATGGCGGTCGT	CGACGCAGGAATGTCAATG
CiOSC	CTTTTGGCCTGGTGACTATGGT	CATTTCCCTTCGATGTTCCACT
CiMYB42	GGGCGAACCGATAACGAGAT	GCTGGAGTCTGGCAGCAAAT
GAPDH	GGAAGGTCAAGATCGGAATCAA	CGTCCCTCTGCAAGATGACTCT

样品 Material	叶长/mm Length of leaf	叶宽/mm Width of leaf	分枝长/mm Branch length	节间数 Number of internodes	节间长/mm Length of internode	茎粗/mm Circumference of stem
WT	33.89 ± 2.07	9.39 ± 0.96	114.81 ± 0.51	7	21.80 ± 4.80	2.74 ± 0.43
OE1	34.10 ± 3.60	10.40 ± 1.22	49.01 ± 0.49^**	6	12.25 ± 2.35^*	2.18 ± 0.33^*
OE2	39.98 ± 0.68^*	11.17 ± 0.93^*	141.46 ± 0.60^**	9	20.68 ± 4.46	2.69 ± 0.41
OE3	34.25 ± 4.39	9.23 ± 1.96	139.10 ± 0.61^**	9	21.19 ± 10.45	2.49 ± 0.23
OE4	33.35 ± 5.16	8.85 ± 0.88	111.51 ± 0.34	7	18.62 ± 3.30	2.45 ± 1.16
OE5	34.54 ± 4.47	9.54 ± 0.84	107.63 ± 0.37^**	8	16.81 ± 6.68	2.40 ± 0.13
OE6	33.43 ± 3.99	8.63 ± 1.07	93.87 ± 0.39^**	7	13.22 ± 2.47^*	1.83 ± 0.25^**
Ri1	33.39 ± 6.14	9.33 ± 0.91	87.31 ± 0.24^**	8	11.78 ± 1.79^*	2.12 ± 0.47^**
Ri2	32.19 ± 6.48	9.63 ± 0.57	61.06 ± 0.33^**	6	10.42 ± 0.55^*	2.13 ± 0.18^*

样品 Material	叶长/mm Length of leaf	叶宽/mm Width of leaf	分枝长/mm Branch length	节间数 Number of internodes	节间长/mm Length of internode	茎粗/mm Circumference of stem
WT	33.89 ± 2.07	9.39 ± 0.96	114.81 ± 0.51	7	21.80 ± 4.80	2.74 ± 0.43
OE1	34.10 ± 3.60	10.40 ± 1.22	49.01 ± 0.49^**	6	12.25 ± 2.35^*	2.18 ± 0.33^*
OE2	39.98 ± 0.68^*	11.17 ± 0.93^*	141.46 ± 0.60^**	9	20.68 ± 4.46	2.69 ± 0.41
OE3	34.25 ± 4.39	9.23 ± 1.96	139.10 ± 0.61^**	9	21.19 ± 10.45	2.49 ± 0.23
OE4	33.35 ± 5.16	8.85 ± 0.88	111.51 ± 0.34	7	18.62 ± 3.30	2.45 ± 1.16
OE5	34.54 ± 4.47	9.54 ± 0.84	107.63 ± 0.37^**	8	16.81 ± 6.68	2.40 ± 0.13
OE6	33.43 ± 3.99	8.63 ± 1.07	93.87 ± 0.39^**	7	13.22 ± 2.47^*	1.83 ± 0.25^**
Ri1	33.39 ± 6.14	9.33 ± 0.91	87.31 ± 0.24^**	8	11.78 ± 1.79^*	2.12 ± 0.47^**
Ri2	32.19 ± 6.48	9.63 ± 0.57	61.06 ± 0.33^**	6	10.42 ± 0.55^*	2.13 ± 0.18^*

材料 Material	气孔密度/（个 · mm^-2） Stomatal density	气孔长/μm Stomatal length	气孔宽/μm Stomatal width	长/宽 Length/width
WT	513.48 ± 67.48	6.89 ± 0.79	2.84 ± 0.47	2.48 ± 0.45
OE1	795.27 ± 172.92^*	8.90 ± 1.73^**	3.85 ± 0.97^*	2.47 ± 0.87
OE4	633.16 ± 43.43	9.08 ± 2.18^**	3.36 ± 0.50^*	2.80 ± 1.00
OE6	590.32 ± 197.93	8.70 ± 1.84^*	3.49 ± 0.33^*	2.51 ± 0.55
Ri1	451.17 ± 78.43	7.45 ± 0.89	3.75 ± 0.53^**	2.03 ± 0.43^*
Ri2	450.78 ± 77.64	7.50 ± 1.03	3.80 ± 0.51^**	2.01 ± 0.42^*