BMC genome

 

Genome-wide screening of the RNase T2 gene family and functional analyses in jujube

BACKGROUND

RNA depolymerase, also known as ribonuclease( RNase), is a type of acidic endonuclease that belongs to the RNase T2 family( 1). In 1959, RNase T2 was linked in Aspergillus fungi( 2). Recent studies showed that S- glycoproteins in tobacco are responsible for tone- incompatibility and are largely homologous to RNase T2 and RNase Rh from Rhizopus niveus(). S- glycoproteins have RNase exertion and RNase T2 family members have two conserved active- point fractions( CASI and II)( 7). These findings indicated that the function of RNase T2 family members is nearly related to factory gametophytic tone- incompatibility, and the RNase T2 genes have been largely conserved throughout elaboration. These discoveries could promote inquiries on the medium of factory tone- incompatibility.

An adding number of RNase T2/ S- RNase enzymes have latterly been discovered in the genomes of shops in Acacia, Solanaceae and Rosaceae( 3,). Among them, RNases were set up in Japanese pear( Pyrus) and were shown to be involved in tone- incompatibility of gametophytes( 11). The S- RNase genes of apricot( Prunus armeniaca) and loquat( Eriobotrya japonica) have also been linked as being related to gametophytic tone- incompatibility( 12, 13). therefore, the RNase T2/ S- RNase enzymes are nearly related to gametophytic tone- incompatibility and should be extensively studied to increase the effectiveness of mongrel parentage. likewise, it has been set up that RNases is involved in abiotic stress responses similar as swab stress, phosphate starvation and anility( 14).

Jujube( Ziziphus jujuba Mill.) a evanescent fruit tree species native to China, has been distributed worldwide(). Low regenerating set and seed product were crucial obstacles for mongrel creation in jujube cross parentage and no mongrel cultivar was successfully employed in civilization( 18). The disquisition of RNase T2 family members and functions will be helpful for jujube cross parentage. still, the work on jujube RNase family has yet not been conducted, and the functions of ZjRNase are still unclear. The end of this study is to identify the RNase T2 gene family in jujube genome and demonstrate the implicit function of RNase T2 genes. The results handed new perceptivity into the molecular mechanisms of low number of cold-blooded seeds in jujube and set up new functions of RNase T2 family members in splint and fruit development

Abstract

BACKGROUND

Ribonuclease( RNase T2) plays pivotal places in factory elaboration and parentage. still, there have been many studies on the RNase T2 gene family in Ziziphus jujubaMill., one of important dried fruit tree species. lately, the released sequences of the reference genome of jujube give a good chance to perform genome-wide identification and characterization of ZjRNase gene family in the jujube

RESULT

In this study, we linked four members of RNase T2 in jujube distributed on three chromosomes and unassembled chromosomes. They all contained two conserved spots( CASI and CASII). Analysis of the phylogenetic connections revealed that the RNase T2 genes in jujube could be divided into two groups ZjRNase1 and ZjRNase2 belonged to class I, while ZjRNase3 and ZjRNase4 belonged to class II. Only ZjRNase1 and ZjRNase2 expression were shown by the jujube fruit transcriptome analysis. So ZjRNase1 and ZjRNase2 were named functional verification by overexpression metamorphosis of Arabidopsis. The overexpression of these two genes led to an roughly 50 reduction in seed number, which earn farther attention. also, the leaves of the ZjRNase1 overexpression transgenic lines were coiled and twisted. Overexpression of ZjRNase2 redounded in docked and crisp siliques and the product of trichomes, and no seeds were produced

Results

Genome-wide identification of RNase T2 family member

Eight RNases have been linked in Oryza sativa( 1). The identification of jujube RNase T2 was performed via BLASTP searches and the Oryza sativa RNases protein sequences were used as query sequences to search the Ziziphus jujuba genome( 19). A total of 4 ZjRNases were linked by two rounds of BLASTP and conserved sphere prognostications( Table 1). The protein length from ZjRNase1 to ZjRNase4 is and 158 and their garbling genes are located on chromosomes Chr1, Chr9, Chr10 and ChrUn, singly. The predicted pIs ranges from5.12 to7.81, and the molecular weight is between18.61 and31.08 kDa. The instability index analysis showed that ZjRNase1, ZjRNase3 and ZjRNase4 are stable, exceptforZjRNase2.The advanced the aliphatic index was, the more stable the protein among the jujube RNase T2 family memberS.
To more determine their evolutionary relationship and grease the type of ZjRNases, a phylogenetic tree was constructed comprising the sequences of 14 MdRNases, 9 PyRNases and 4 ZjRNases(Fig. 1). The 4 ZjRNases were divided into two types class I and class II(Fig. 1). ZjRNase1and ZjRNase2belongs to class I groups I a and I b, ZjRNase3 and ZjRNase4 belong to class II
previously reported RNases have two conserved active spots, CASI and CASII motifs( 20). All the ZjRNases have the same conserved structural spots as the RNases in other plant species, suggesting that the four ZjRNase members we attained in jujube are correct(Fig. 2). According to secondary structure analysis, it was predicted that α- helix or β- distance structures are present at 14 positions, of which 7( 50) may be α- helix structures( red) and 5(35.71) may be β- distance structures( light green). The remaining two prognostications were inconsistent and were classified as uncertain types. These results showed that the secondary structure of the ZjRNase member proteins are fairly stable

Discussion

Ribonuclease( RNase T2) play pivotal places in factory elaboration and parentage and an adding number of RNase T2/ S- RNase enzymes have latterly been reported in genome of different shops, similar as Acacia, Solanaceae and Rosaceae( 3,). Through genome-wide mining, four members of the RNase T2 family were linked in jujube. These members could be divided into two orders according to phylogenetic analysis, which is fairly low compared with that of other species( 1, 5).

The former study showed that RNase T2 is related to tone- incompatibility and abiotic stress responses in shops( 1, 3, 5). Some genes involved in seed and fruit development have been reported( 21, 22). For case, YABBY 、 OVATE and EPFL2 told fruit size and shape, ORANGE and MPK4 were related to seed number(). still, the exploration about RNase T2 function during fruit and seed development was absent. In terms of bioinformatics, the RNase T2 gene family members in jujube have characteristics analogous to those of RNases of other factory species( 1, 5). In particular, the jujube RNase T2 gene retains the original capability of T2 genes, and when overexpressed in Arabidopsis, these genes could lead to reduced seed product. The VvNAC26 transgenic shops were set up to reduce tomato seeds( 30) and INSENSITIVE2( EIN2) encodes a membrane protein and affected seed development( 31). This study showed that ZjRNase1 and ZjRNase2 had commerce with NAC and EIN2, independently. It indicated NAC and EIN2 recap factors were presumably associated with function of ZjRNase1 and ZjRNase2 involved in seed development.

In addition, the jujube RNase T2 family members showed some differences in terms of their function. The class I member ZjRNase1, after being overexpressed, was set up to be largely expressed only in flowers and caused a crooked- splint phenotype in addition to siliques dock and reduced seed number. Analysis of its protagonist revealed that the sequence( CAAT( A/ T) ATTG) may share in the isolation of the palisade towel and can also bind HD- ZIPI recap factors, which have been shown to play a part in splint morphogenesis( 32). In addition, HD- ZIPI proteins were linked as able of interacting with RTNLB8 and VAP27- 1 in terms of protein relations( 33). All of these interacting proteins are largely expressed during splint morphological development. The class II member ZjRNase2 was expressed in the flowers of the transgenic lines. Overexpression of this gene caused crisp siliques, reduced seed figures and indeed no seeds, as well as the product of trichomes. According to the literature, shops produce further trichomes to repel pests, indicating that similar gene may also be associated with nonentity resistance

Materials and methods

Plant materials and cultivation

Arabidopsis thaliana( gap- 0) seeds were attained from Xuan Zhao from China Agriculture University and sown in a soil medium matrix( peat vermiculite = 11) under a 16 h light/ 8 h darkness photoperiod at 20 ± 2 °C and a relative moisture of 60 ± 5. The seeds of Arabidopsis thaliana( gap- 0) shops were grown on 1/ 2- strength Murashige and Skoog( MS) media. All the shops were grown under the same conditions. RNA was uprooted using a TIANGEN factory RNAprep Pure Factory tackle DP432( TIANGEN biotech company). Jujube fruit transcriptome data were attained from the National Center for Biotechnology Information( NCBI) database

Database quests and identification of RNase genes in the Ziziphus jujuba genome
The genome sequences of Ziziphus jujuba Mill. were attained from the National Center for Biotechnology Information( NCBI) database( 19). The sequences of 14, 9 linked MdRNases, PyRNases were downloaded from the National Center for Biotechnology Information( NCBI) database. In addition, the sequences of8 linked OsRNases were downloaded( 1). RNases were linked by two rounds of BLASTP quests. First, the sequences of all OsRNases were used to search for possible ZjRNases sequences via TBtools( 36). also, NCBI Batch CD- Hunt was used to confirm whether the seeker RNases contained theRNase_T2 superfamily sphere( pfam00445) or theRibonuclease_T2 sphere( cl00208). A aggregate of 4 ZjRNase genes were eventually linked in the genome. The protein length, isoelectric point( pI) and molecular weight( MW) were latterly prognosticated.

Phylogenetic analysis and multiple sequence alignment
Sequences of the OsRNase proteins were attained from the Phytozome database. A neighbour- joining( NJ) phylogenetic tree comprising the full- length sequences of MdRNases, PyRNases and ZjRNases was constructed with 1000 bootstrap replicates using MEGA7.0. Multiple sequence alignment of all ZjRNases was also performed by MEGA7.0.

RNase gene structure and conserved motif analysis
The RNase gene structure and conserved disciplines were analysed and imaged using TBtools software( 36). The conserved motifs of the linked ZjRNase proteins were explored with the help of the Multiple Anticipation Maximization for Motif Elicitation( MEME) online program.

Analysis of cis- acting rudiments of ZjRNases
The implicit nonsupervisory cis- acting rudiments of jujube RNases were checked by using TBtools software; the region 2000 bp upstream of the launch codon was estimated. also, the cis- acting rudiments in the protagonist were prognosticated via PlantCARE online software to identify their nonsupervisory functions

CONCLUSION

In this study, four ZjRNase genes and their corresponding protein sequences were linked from the jujube genome, and the ZjRNase genes were divided into two orders. Class I had member ZjRNase1 and ZjRNase2, ZjRNase3 and ZjRNase4 belonged to Class II but not expressed in the fruit. thus, ZjRNase1 and ZjRNase2 were named for functional verification. As a result of the transgene, we innovated that ZjRNase1 and ZjRNase2 could lead to a drop in the number of seeds, inferring that ZjRNase genes might be involved in the conformation of seeds. This study provides a base for farther studies on the functional parcels of RNase genes; still, farther studies are demanded to more interpret the nonsupervisory medium of these four RNase genes on seed conformation in jujube parentage