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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (3): 597-607.doi: 10.3724/SP.J.1006.2023.22014

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Identification of sheathed panicle mutant sui1-5 and screening of OsPSS1 interaction protein in rice (Oryza sativa L.)

YANG Ye1(), SUN Qi2, XING Xin-Xin2, ZHANG Hai-Tao1,*(), ZHAO Zhi-Chao2,*(), CHENG Zhi-Jun2   

  1. 1College of Agronomy, Anhui Agricultural University, Hefei 230036, Anhui, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2022-03-05 Accepted:2022-07-21 Online:2023-03-12 Published:2022-08-19
  • Contact: ZHANG Hai-Tao,ZHAO Zhi-Chao E-mail:874498704@qq.com;43647174@qq.com;zhaozhichao@caas.cn
  • Supported by:
    National Natural Science Foundation of China(31871603)

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Abstract:

The sheathed panicle phenomenon is a disadvantageous trait in agricultural production, which is mainly due to the abnormal elongation of the uppermost internode, resulting in the panicle closed by flag leaf sheath. Although some mutants of sheathed panicle have been identified and reported in rice, the underling molecular mechanism of uppermost internode shortening is still poorly understood. OsPSS1/SUI1 is a phosphatidylserine synthase gene and the mutation of OsPSS1 leads to a severe shortening of the uppermost internode of mutant sui1-4. We discovered and identified a new allelic mutant sui1-5 of SUI1 and the degree of the uppermost internode shortening in sui1-5 was significantly reduced. Sequence analysis revealed a single nucleotide substitution of G to T at the seventh exon of SUI1 in sui1-5, resulting in an amino acid change from glycine to valine. To study the function of OsPSS1, we detected a protein GH9A3 interacting with SUI1 through a yeast screening library, which was verified by yeast two-hybrid, luciferase complementarity (LCI), and bimolecular fluorescence complementarity (BiFC). GH9A3 was a member of GH9 gene family, encoding an endo-β-1,4-glucanase which was speculated to be a key enzyme in cell wall cellulose synthesis. At seedling stage, the relative expression level of SUI1 was not synchronized with that of interaction protein-coding gene GH9A3, and the relative expression level of GH9A3 was up-regulated in sui1-5. But at heading and jointing stage, the relative expression level of SUI1 and GH9A3 decreased synchronously, and the relative expression level of other cellulose synthase CESAs family genes decreased obviously in sui1-5. These results lay a foundation for further study of the function of OsPSS1.

Key words: sheathed panicle, SUI1, GH9A3, fine mapping, cellulose synthesis

Table 1

Indel and SNP primers for fine mapping"

多态性分子标记
Polymorphic marker		 正向引物
Forward sequence (5'-3')		 反应引物
Reverse sequence (5'-3')
Inc1-1 AGGGGAAGAAAAACCTGACC CCGCGTGCAGATAAAGTACA
Y1-1 CAAGCAGTGATCATACAGCCTTCC GCCATGGCTGAGAACAGAGAGC
Y1-2 CTCCTGTTACAACTGCATGGA TCGATCCATTAGCTAGCCGG
Y1-3 CATGAGTACAACATCGCCTACA CGATGATTCAGTACATGCATGC
Y1-4 GCTGCATCGGTATACAAGACG TCAGTTTGTCAGGGAGGGAG
Y1-5 CTCCGTTCTCCACCGTGT AACAGCCAACTAGGTTCACA
Y1-6 GCGGCCACCATGATCTTGTACC GTACTTGTTGTAGCGCCCGTTCC
Y1-7 CTAGCAGCTGTCTGCGACACACG CCGAGGTGTTATGCCAATCTCTATGG

Table 2

Primers used for qRT-PCR"

基因名称
Gene name		 正向引物
Forward sequence (5'-3')		 反应引物
Reverse sequence (5'-3')
OsCesA1 TCTTCTCGCCACGAACAACA CTTGGAGGGGTCCACAATCC
OsCesA3 TGCCAGCCATCTGTTTGCTA GCCAACACCACTCCATCTCA
OsCesA4 AAGGGATCAGCGCCAATCAA GGAGCCATTTCAGACGACCA
OsCesA7 TGGAAGTCGGTGTACTGCAC GCGGCTCATGAAGATCTCGA
OsCesA8 ATGACCAACGGGACAAGCAT TAGAGAGAGGCTGGCGAGTT
OsCesA9 GCGCCGATCAACCTATCTGA CTTGTAGCCGTAGAGGAGCG
OsGH9A3 TGAGTGCCACTCTTGCTCAG CCGAACTTGTACAGGGCCTT
SUI1 GCAGACCCGTGATGATGCTA TATATGCGGCAGTCGGTTCC
Ubiquitin ACCACTTCGACCGCCACTACT ACGCCTAAGCCTGCTGGTT

Fig. 1

Phenotypic comparison between wild type and mutant sui1-5 A: phenotypes of Kitaake (left) and sui1-4 (right) plants at heading stage; B: phenotypes of Kitaake (left) and sui1-5 (right) plants at heading stage; C: phenotype comparison of the sheathed panicle and the internode length of WT (left) and sui1-5 (right) at heading stag; D: contribution rate of internode length to plant height of wild type and mutant; E, F: longitudinal sections of the second internode of wild type (E) and sui1-5 (F); G, H: comparisons of the length and width of stem parenchyma cells from longitudinal sections of the second internode in wild type and sui1-5; I, J: transverse sections of the second internode of wild type (I) and sui1-5 (J); K: the comparison of cell numbers of the radius in the transverse sections of the second internode in wild type and sui1-5. Bar: (A) 10 cm, (B) 10 cm, (C) 10 cm, (E, F) 100 μm, (I, J) 100 μm. * represents significantly different at P < 0.05."

Table 3

Agronomic traits comparison between wild type and mutant"

农艺性状
Agronomic trait		 野生型
Kitaake		 突变体
sui1-5
株高Plant height (cm) 78.90±4.80 68.80±5.40**
分蘖数Tiller number 23.40±2.37 31.50±3.30**
剑叶宽Flag leaf length (cm) 1.21±0.09 1.08±0.08**
一次枝梗Primary branch number 6.80±0.92 8.30±1.50*
二次枝梗Secondary branch number 7.50±1.96 1.70±1.25**
穗长 panicle length (cm) 14.09±0.96 9.27±1.01**
倒一节间长First internode length (cm) 26.64±3.47 0.21±0.12**
倒二节间长Second internode length (cm) 17.51±0.98 14.18±0.88**
倒三节间长Third internode length (cm) 9.35±1.19 8.63±1.58
倒四节间长Fourth internode length (cm) 2.53±0.37 1.79±0.51**
每穗粒数Grain number per panicle 67.10±8.78 29.80±4.64**
结实率Seed setting rate (%) 96.00±0.03 62.00±0.06**
千粒重1000-grain weight (g) 26.13±0.04 26.89±0.06**
粒长Seed length (mm) 6.79±0.15 6.87±0.19
粒宽Seed weight (mm) 3.33±0.04 3.39±0.13
粒厚Seed thickness (mm) 2.20±0.05 2.34±0.32

Fig. 2

Map based cloning of gene SUI1 A: the primary mapping of SUI1 between markers Y1-1 and Y1-7 on the 1st chromosome; B: SUI1 was fine mapped to the interval between the markers Y1-3 and Y1-4, a 38.05 kb region. Above the line is the molecular marker used for gene mapping, and below the line is the recombinants identified by the corresponding markers. C: 3 ORFs within the fine-mapped region; D: the SUI1 gene contains 12 exons and 11 introns. The white block represents the coding region and the black block represents the non-coding region. The line represents the sequence comparison of Kitaake and sui1-5 in the seventh exon, and the arrow points to the mutant base."

Fig. 3

Interaction validation of SUI1 and GH9A3 proteins A: the interaction between SUI1 and GH9A3 in a yeast two-hybrid system. AD: activation domain; BD: DNA binding domain; SD: nutrient deficient medium. B: LCI assay indicates that SUI1 interacts with GH9A3 in N. benthamian leaves. C: the interaction validation between SUI1 and GH9A3 by BiFC assay in N. benthamiana, mCherry: SCAMP1. At least three independent N. benthamiana leaves was repeated."

Fig. 4

Phylogenetic tree of OsGH9A3 protein in rice The OsGH9A3 protein sequence was used as a Qurry sequence, the candidate species including Triticum aestivum, Zea mays, and Arabidopsis thaliana, and the homologous protein obtained by blast were constructed the evolutionary tree with Neighbor-Joining method in MEGA 7.0. The boot program value is shown as a percentage on the node. The branch length is used to infer the evolutionary distance of phylogenetic trees. OsGH9A3 had the closest homology with gpm617 protein in Zea mays, and the homology between rice and Arabidopsis GH9A family proteins was also relatively high."

Fig. 5

Subcellular localization of GH9A3 in tobacco epidermis The above figure shows the control of 35S-GFP empty carrier. The following figure shows the 35S-GFP vector connecting GH9A3 fragment. mCherry: AtSYP122, GH9A3 is located on the plasma membrane of tobacco epidermal cells with dot structure."

Fig. 6

Relative expression levels of SUI1 and GH9A3 was significantly reduced in mutants at heading stage A: seedling phenotype of wild type and mutant sui1-5, left: wild type, right: sui1-5; B: the expression changes of SUI1 and GH9A3 in sui1-5 at seeding stage (left) and heading stage (right). The relative expression levels of two genes in the mutant were decreased significantly at heading stage. **: P < 0.01."

Fig. 7

Relative expression level of GH9A3 and CESAs family genes The relative expression level of CESAs was significantly down- regulated in sui1-5 culms at heading stage. OsCesA1, OsCESA3, and OsCESA8 are involved in primary cell wall cellulose biosynthesis. OsCESA4, OsCESA7, and OsCESA9 are responsible for cellulose biosynthesis in the secondary cell walls. **: P < 0.01."

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