Interaction Identification Between mRNA Splicing Factor TaSR and TaHis as well as Expression Analysis of TaSR in Wheat

doi:10.7668/hbnxb.20194686

Abstract

Abstract:

To study the resistance mechanism of TaHis gene to Fusarium head blight(FHB)in wheat,the full-length coding sequence of TaHis was cloned,and the bait vector pGBKT7-TaHis was constructed,which was then used as bait for screening a yeast two-hybrid library of wheat ear induced by FHB.After obtaining the interacting proteins,yeast two-hybridization and bimolecular fluorescence complementation were further used to verify the interaction between these proteins,and RT-qPCR was used to analyze the expression pattern of TaHis interacting protein induced by FHB in resistant and susceptible cultivars respectively.The results showed that the bait vector pGBKT7-TaHis was successfully constructed,and 18 yeast monoclones were obtained on the four deficient selection medium(SD/-Leu/-Trp/-His/-Ade)after yeast two-hybrid library screening.Blast analysis showed that a total of 5 proteins were obtained,and the coding sequence of serine/arginine-rich mRNA splicing factor SR45a-like(TaSR)was identified in 6 colonies.We cloned the full-length coding region of TaSR gene from Bainong 4299 and constructed pGADT7-TaSR vector.The experiment of yeast two-hybrid showed that the yeast cells co-transformed with pGADT7-TaSR and pGBKT7-TaHis grew well and appeared blue on SD/-Leu/-Trp/-His/-Ade/ X-α-Gal/AbA,indicating that TaSR and TaHis directly interacted in yeast cells.The vectors of YC-TaHis and YN-TaSR were constructed,and the bimolecular fluorescence complementary experiments were performed.The results showed that strong fluorescence signals were generated in tobacco cells co-transferred with YC-TaHis and YN-TaSR,which further verified the interaction between TaSR and TaHis.RT-qPCR analysis of TaSR gene expression showed that TaSR expression was up-regulated in resistant cultivar Bainong 4299,while down-regulated in susceptible cultivar Bainong 607 upon FHB infection,indicating a positive correlation between TaSR expression level and FHB resistance in wheat.To sum up,the interaction between wheat TaSR and TaHis was proved,and TaSR expression level was positively correlated with FHB resistance in wheat.

Key words: Fusarium head blight, TaHis, TaSR, Yeast two-hybrid, Bimolecular fluorescence complementation, RT-qPCR

SONG Puwen, DENG Jiale, DU Yuxin, CHEN Jiamei, JING Yueting, LIU Juntong, LI Ao, HU Haiyan. Interaction Identification Between mRNA Splicing Factor TaSR and TaHis as well as Expression Analysis of TaSR in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 166-172. doi: 10.7668/hbnxb.20194686.

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http://www.hbnxb.net/EN/Y2024/V39/I3/166

Figures/Tables 8

Tab.1 Primer sets used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
TaSR-F	ATGGCGCCGTCCGCCAG
TaSR-R	TCAATCATAACTGGCATTCAGAAAC
TaHis-F	ATGTGTATATTTGGACAACCATGTA
TaHis-R	TTACACGAGTTGCTTCCCGT
AD-TaSR-F	GGAGGCCAGTGAATTCATGGCGCCGTCCGCCAG
AD-TaSR-R	CGAGCTCGATGGATCCTCAATCATAACTGGCATTCAGAAAC
BD-TaHis-F	CATGGAGGCCGAATTCATGTGTATATTTGGACAACCATGTA
BD-TaHis-R	GCAGGTCGACGGATCCTTACACGAGTTGCTTCCCGT
BiFC-TaSR-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGCCGTCCGCCAG
BiFC-TaSR-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCATCATAACTGGCATTCAGAAAC
BiFC-TaHis-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTGTATATTTGGACAACCATGTA
BiFC-TaHis-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCCACGAGTTGCTTCCCGTC
qTubulin-F	ATCTCCAACTCCACCAGTGTCG
qTubulin-R	TCATCGCCCTCATCACCGTC
qTaSR-F	AGCTTGAGAAATCCAAGGAAGA
qTaSR-R	CCACTTTCGACAAGTTGTCAAT

Fig.1 Cloning of TaHis gene and construction of pGBKT7-TaHis bait vector A.PCR amplification of TaHis gene;B.Identification of bait vector pGBKT7-TaHis by double enzyme digestion.M.DNA Marker DL2000+.

Tab.2 TaHis interacting protein statistics

蛋白质ID Protein ID	蛋白质名称 Protein name	酵母克隆数量/个 Number of yeast clones
XP_037440604.1	小麦CAX相互作用蛋白	2
XP_037468224.1	丝氨酸苏氨酸蛋白激酶RIO1	3
XP_044367519.1	富含丝氨酸/精氨酸的mRNA剪接因子SR45a类蛋白	6
XP_044437442.1	泛素受体RAD23b	3
XP_044369301.1	二磷酸核酮糖羧化酶/加氧酶活化酶A	4

Fig.2 Cloning of TaSR gene and construction of pGADT7-TaSR vector A.PCR amplification of TaSR gene;B.Identification of pGADT7-TaSR vector by double enzyme digestion.M.DNA Marker DL2000+.

Fig.3 PCR detection of bimolecular fluorescent complementary vectors for TaHis and TaSR genes A.PCR detection of YC-TaHis vector;B.PCR detection of YN-TaSR vector.M.DNA Marker DL2000+.

Fig.4 Interaction between TaSR and TaHis detected by yeast two-hybrid system

Fig.5 Interaction between TaHis and TaSR detected by bimolecular fluorescent complementation

Fig.6 Quantitative PCR analysis of TaSR expression after F.graminearum infection Different capital letters indicate significant differences(P<0.05)between different cultivars at the same time point,and different lowercase letters indicate significant difference(P<0.05)between different time points for the same cultivar.

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