Analysis of GhSuSy expression by particle bombardment during initiation of cotton fibers

doi:10.3969/j.issn.1004-1524.2022.07.02

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (7): 1361-1368.DOI: 10.3969/j.issn.1004-1524.2022.07.02

• Crop Science • Previous Articles Next Articles

Analysis of GhSuSy expression by particle bombardment during initiation of cotton fibers

LYU Shufang(), ZHANG Hongxiao, XU Huawei, ZHAO Xingli

College of Agriculture, Henan University of Science and Technology, Luoyang 471003, Henan, China

Received:2021-09-23 Online:2022-07-25 Published:2022-07-26

Abstract

Abstract:

Sucrose synthase mediated the initiation and development of cotton fiber through regulating plant sugar metabolism. In order to further research the transcription mechanism of sucrose synthase in the process of the cotton fiber initial, the 2 000 bp promoter fragment of GhSuSy gene upstream was cloned and recombinant vector with luciferase (LUC) fluorescent reporter gene was constructed. The recombinant vector DNA were transformed into cotton leaves and petals by particle bombardment, respectively, and LUC activity were compared and analyzed under different pressure, different bombarding distance and incubation time.LUC activity in leaves and petals were determined at the initial differentiation stage of cotton fibers and were compared with GhSuSy expression and sucrose synthase activity. The results showed that the conversion efficiency was the highest under vacuum degree of -0.09 MPa, the pressure was set as 6.80 MPa, the distance of carrier film from the blade was 7 cm after two times of bombardment, LUC fluorescence was the strongest after the transformed samples were incubated for 16 h. Compared with LUC in leaf, LUC fluorescence in petal were stronger and activity were higher, especially at 0 and 1 DPA(days post anthesis).From 3 days before flowering (-3 DPA) to 3 days after flowering (3 DPA), the LUC activity in leaves and petals increased first and then decreased, and the LUC activity reached the highest at 0 and 1 DPA, which were consistent with the quantitative analysis of GhSuSy and sucrose synthase activity in leaves, petals and ovules. These results suggested that GhSuSy gene or the sucrose synthase significantly regulated the initiation and differentiation of cotton fibers at 0 and 1 DPA, and showed that gene gun-mediated transient transformation system could be used to analyze the transcriptional regulation of GhSuSy gene promoters.

Key words: cotton fiber, GhSuSy gene, sucrose synthase, particle bombardment

CLC Number:

S562

LYU Shufang, ZHANG Hongxiao, XU Huawei, ZHAO Xingli. Analysis of GhSuSy expression by particle bombardment during initiation of cotton fibers[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1361-1368.

Figures/Tables 8

Table 1 Information of qRT-PCR primers

基因Gene	上游引物Forward primer(5'→3')	下游引物Reverse primer(5'→3')	产物长度Production length/bp
GhUBQ7	GAAGGCATTCCACCTGACCAAC	CTTGACCTTCTTCTTCTTGTGCTTG	167
GhSuSy	TCGTAGTTGTAGGTGGTGATAGG	CACCGTTACAGGTTGCGAATG	275

Fig.1 Construction of recombinant vector A, Promoter fragment of GhSuSy; B, Transient expression vector.

Table 2 Effects of parameters of particle bombardment on activity of LUC

可裂膜压力 Pressure/ MPa	载体膜到叶片不同距离(cm)的LUC荧光强度 Luorescence intensity of luciferase of different target distance(cm)
可裂膜压力 Pressure/ MPa	5	7	9
4.60	(2.84±0.47) ×10³	(2.06±0.45) ×10³	(1.18±0.33) ×10³
6.80	(0.86±0.29) ×10⁵	(1.28±0.61) ×10⁵	(0.63±0.25) ×10⁵
9.20	(0.24±0.15) ×10³	(3.96±1.64) ×10³	(3.88±1.41) ×10³

Fig.2 Effects of the times of particle bombardment on LUC activity

Fig.3 Effects of incubating time on LUC activity

Fig.4 Relative luciferase activity in leaves and petals during the initiation of fiber development

Fig.5 Expression of GhSuSy in leaves, petals and ovules during initiation of fiber development

Fig.6 Sucrose synthase activity in leaves, petals and ovules during initiation of fiber development Data was detected based on fresh weight.

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Analysis of GhSuSy expression by particle bombardment during initiation of cotton fibers

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