Cloning And Cold Resistance Analysis Of β-1,3 Glucanase Gene (Mugsps) From Wild Banana

Musa genus plants (Family:Musaeae, Musa spp.)are important foliage plants of giant tropical and subtropical monocotoyledenous herbs which are widely used in various types of garden greening. Planting area are limited due to Musa genus plants are vulnerable to low temperature damage. The damages such as leaves etiolation, withering and water-soaking are caused by cold damage, reducing the ornamental value of Musa genus plants. Therefore, it is important to improve the cold resistance of Musa genus plants. In this study, we reported the low semi-lethal temperature of ten banana plant germplasms, exploring suitable planting area of different Musa genus plants. Research was conducted to study the cold resistant molecular mechanism of Sanming wild banana including determination of the conductivity changes and β-1,3 glucanase activity changes under low temperature. Functional and evolutionary analysis of β-1,3 glucanase (GSP) gene family, cloning of cold resistance related gene Mugsps, subcellular localization analysis and real-time fluorescence quantification (qRT-PCR) expression analysis of Mugsps were also performed.1 Physiological and biochemical study under low temperature of different genus Musa genus plants1.1 Electrolytic leakage rate measurement of the genus Musa genus plants under low temperatureTo compare the cold resistant capacity among different Musa genus plants, relative electric conductivity of 10 kinds of Musa genus plants were measured and low semi-lethal temperature (LT50) were calculated, then we measured the lowest temperature of Sanming wild banana can resistant.(T)We picked leaves from 10 kinds of banana plant for material, using logistic equation calculated LT50.Afer 7 different temperatures treatments (28℃,1℃,0℃,-1℃,-2℃,-3℃,-4℃ for 36 h), the increase of relative electric conductivity showed curve of single "S" pattern, and the LT50 trendencies of different kinds of bananas were Sanguinea banana (-0.377 ℃)< Beijiao banana (-0.477℃)< Tianbao plantian (-0.739℃)< Fuzhou wild banana (-1.086℃)< Local banana(-1.187℃)< Chaijiao banana(-1.204℃)< Bangladesh banana(-1.307℃)< Sandiejing banana (-1.389℃)< Australia banana (-1.559℃)< Sanming wild banana (-1.776℃).The results indicated that the cold resistance of wild bananas was stronger than cultivated Musa genus plants. The study provided references for scientific planting of Musa genus plants.② The relative electric conductivity of detached new leaves from Sanming wild banana was measured under 0℃ of 0 h,1 h,4 h,8 h,12 h,24 h,5 d,10 d,15 d treatments. The results indicated that along with the temperature decreasing, the electrolyte leakage showed wavy upward trend, after treatment for 4 h and 24 h, the electrolyte leakage decreased to 23.68%(lower than control group 24.81%) and 25.34%, respectively. This result described that under the condition of tests the leaves of Sanming wild banana might experienced two cold response processes i.e the membrane structure was protected instantly for the first time of cell cold responded at 4 h, with the treatment time of low temperature prolonged, cell were destroyed, and the second time of cold responded was happened which generated the antifreeze proteins, protective enzyme, etc. at 24 h. After a period of exposure to low temperature (5 d) through a complex adaptive process, the electrolyte leakage could maintain at 27% which suggested that Sanming wild banana acquire increased frost tolerance at 0℃.1.2 β-1,3 glucanase activity determination of Sanming wild banana under different temperature treatmentIn order to determinate the enzyme activities of β-1,3 glucanase in leaves from Sanming wild banana under different cold stresses (13℃,8℃,4℃,0℃,-2℃,-4℃,-6℃ for 36 h). The crude enzyme extract was reacted with laminarin and the reducing sugar products were measured by 3,5-dinitrosalicylic acid (DNS) method. We defined that produce 1 μmol/min of reducing sugar required amount of β-1,3 glucanase as a enzyme activity unit. Results showed that the activities of β-1,3 glucanase showed "descending-ascending-descending" trends that activities up to 1.2 times and 1.4 times of control group at 13℃ (critical low temperature) and -2℃ (low semi-lethal temperature) of Sanming wild banana treatment, respectively. The activities of P-1,3 glucanase under cold treatments indicated that β-1.3 glucanase was stimulated to improve the cold tolerance of the plants.2 Function and evolution analysis of GSP whole genes family based on genome databasePlant β-1,3 glucanase (EC.3.2.1.39) belongs to the glycosyl hydrolase enzyme family 17 which has many members and complex functions. We identified all genes for proteins containing GSP domain (Pfam:PF00332) by the HMMER program (2.3.2) from local genomic databases of bananas (M. acuminata and M. balbisiana), obtained 65 GSPs (removed 30 sequences of without GSP domain and add 2 new sequences) and 69 GSPs (removed 23 sequences of without GSP domain and add 9 new sequences) from M. acuminata and M. balbisiana genomic databases, respectively. The M. acuminata and M. balbisiana GSP family could also be classified in to three subfamilies of α(α1,α2,α3),β (β1,β2,β3) and γ referred to the classifications of Arabidopsis, based on the phylogenetic analysis of GSP family of the M. acuminata and Arabidopsis. The functions of a and y mainly involved in plant growth and development while β most likely has PR-proteins function. The phylogenetic tree of M. acuminata and M. balbisiana showed that besides the gene named "ITC1587 Bchr2 P03966" from M. balbisiana, had low similarity with other GSP members, was classified as new subfamily of δ, others had high homology. The analysis of GSP family from M. acuminata showed that the GSP family genes were located in 2 to 11 chromosome, without tandemm repeats, so that whole family genetic replication might be the main way of GSP gene family amplification. According to the calculated value of Ka/Ks which was less than 1, we assumed that gene purify selection plays a major role after experienced the three times of whole family genetic replication events of Musa genus plants. Through the above analysis, we also suggested that the reason of β subfamily genes have PR function might due to the chromosomes integrated with the foreign PR gene segments during evolution.3 Cold resistance related gene Mugsps clone from wild bananaFull ORF length cDNA and DNA sequences of β-1,3 glucanase genes (Mugspl.2, Mugsp2, Mugsp3, Mugsp4, Mugsp5)were cloned from Sanming wild banana leaves under 13 ℃ treatment for 36 h. Sequence analysis showed that, Mugsp1.2, Mugsp2, Mugsp3, Mugsp4, Mugsp5 all belonged to β1 class of β-1,3 glucanase genes. ORF lengths of cDNA were 1020,1047,999,1023 and 960 bp, encoded 339,348,332,340 and 319 proteins, respectively. Genetic structure analysis showed that Mugsp1.2, Mugsp(915 bp),Mugspl(951 bp)were transcribed from same DNA which Mugsp and Mugsp1 cloned before were exon-lacking alternative splicing genes. Different between the homologous sequences of Mugsps from M. acuminata and M. balbisiana contained many introns; Mugsp1.2, Mugsp2, Mugsp4 contain one intron while Mugsp3 and Mugsp5 did not have intron construction. Bioinformatics predictions indicated that Mugsp1.2, Mugsp4 might Ⅱ class of transmembrane protein encoding basic β-1,3 glucanase with one transmembrane domain, Mugsp2, Mugsp3, Mugsp5 might peripheral membrane proteins encoding acidic β-1,3 glucanase. All Mugsps (Mugspl.2-Mugsp5) have multiple phosphorylation sites include MAPK, without X8, GPI-anchor, CTPP, NLS domain, suggested that Mugsps might belong to new class of PR-related P-1,3 glucanase genes and involved in MAPK cold regulation pathway.4 Subcellular localization analysis of Mugsps from wild bananaSubcellular localization analysis was conducted by constructing the expression vectors of Mugsp1.2, Mugsp2, Mugsp4 with pCAMBIA1302-GFP plasmid and did transient transformation of the genes in onion epidermal cells by Agrobacterium tumefaciens mediation at room temperature. The results of subcellular localization showed that Mugsp1.2-GFP, Mugsp2-GFP, Mugsp4-GFP were expressed on vacuole membrane, plasma membrane and cytoplasm, respectively. Once all Mugspl.2-GFP, Mugsp2-GFP, Mugsp4-GFP could transfer to cell nucleus after room temperature treatment the transformed onion epidermal cells were cultured at 8℃. Before observation undrer microscope, onion epidermal cells were dyed by using 1 μg/mL DAPI. Based on the experimental results, we assumed that Mugsp1.2, Mugsp2, Mugsp4 might carried cold signal to the nucleus by combined certain proteins or phosphorylation under low temperature.5 qRT-PCR expression analysis of Mugsps from wild banana with low temperature treatmentUsing 18s rRNA as the reference gene, the expression levels of Mugsps (Mugsp, Mugsp1, Mugspl.2, Mugsp1, Mugsp3, Mugsp4, Mugsp5) under low temperature treatment were detected by qRT-PCR. The results revealed that:All Mugsps could response to cold stress (28℃,20℃,13℃,4℃,0℃,-2℃,-4℃,-6℃ treated for 36 h), but the expression levels were differed from each other. The expression of Mugsp2 increased firstly then decreased after 4℃, expression increased about 3.5 times of the normal temperature (28℃). Mugsp, Mugspl, Mugsp1.2, Mugsp3, Mugsp4, Mugsp5 showed "M" type expression trends in which Mugspl, Mugsp3, Mugsp4 reached the peak expression at 13℃ and 4℃ about 1.7,2.1,0.8 and 2.5,2.2,1.4 times that of normal temperature, while Mugsp], Mugsp3, Mugsp4 reached the peak expression at 13℃ and 0℃ about 2.0,0.8,0.6 and 2.7,5.0, 4.5 times that of normal temperature, respectively. As the expressions of Mugsp], Mugsp1.2, Mugsp3 higher and responded to low temperature more rapidly, they might play more important role in cold-resistant reaction. qRT-PCR expressions of Mugsps consistent with P-1,3 glucanase activities under low temperature show that Mugsps would translated to β-1,3 glucanase proteins to improve the abilities of resistance and adaptation of bananas to coldness.Under treatments of 8℃ for 1 h,4 h,8 h,12 h,24 h, all Mugsps expressions could improve quickly of which Mugsp, Mugsp], Mugsp1.2 and Mugsp, Mugsp1, Mugsp1.2 reached the highest expression at 1 h and 4 h, respectively. However the expressions of Mugsps were delayed after 0.5 mmol/L SA spray treatment under 8℃ caused Mugsp, Mugsp1, Mugsp1.2 reached the highest expression at 12 h while Mugsp2, Mugsp4, Mugsp5 began to rise. We suggested that SA treatment protected the cell membrane against cold injury so that Mugsps delayed expression for failing to feel changes of membrane. According to the results of qRT-PCR, subcellular localization and bioinformatics analysis of Mugsps, we speculated that one of the functions of Mugsps might be a cold signal transducer, which can rapidly increase gene expression in 1～4 h, through the MAPK signal pathway translate from cell membrane to the nucleus to control the activity of cold related transcription factors under 8℃ temperature. Our experimental results laid a foundation to improve the cold resistance of Musa genus plants and study the mechanism of cold resistance of plants.