| Cotton(Gossypium spp.)is an important economic crop and the largest source of textile fiber in the world.The leaves are the main places for photosynthesis.Many factors affect the cotton yield and the fiber quality such as photosynthetic efficiency,pests and diseases.The morphology of cotton leaves varies considerably.Phenotypes include okra,sea-island,super okra,and broad leaf which are controlled by a multiple allele locus,L2.Okra leaf(L20)is an incomplete mutation that alters leaf shape by increasing the length of lobes with deeper sinuses.Cotton with the okra leaf phenotype has a potential resistance to insect pests and drought.The predominant form in cultivated cottons is the broad leaf type.IL-15-5-1 was derived from a CSIL with sea-island leaves that carried the Hai7124 segment from chromosome D1 in the background of the recurrent parent TM-1.IL-15-5-1 was crossed as a male parent with TM-1 to produce a mapping population(IL-15-5-1 ×TM-1)F2.The L2e locus was mapped in the regin between markers JESPR152 and NAU3040 using simple sequence repeat(SSR)markers.The L2e gene was delimited in a 183-kb region flanked by markers JESPR152 and H1776,based on the genome sequencesof G.raimondii.In the 183-kb region,18 open reading frames(ORFs)were predicted.Quantitative real time PCR(qPCR)of ORFs revealed that the expression of ORF17 was consistently different between the super okra leaf(Super okra),okra leaf(T586),sea-island leaf(Hai7124)and broad leaf(TM-1).This gene was not expressed detectably in the broad leaf(TM-1),but its expression was significantly higher in the super okra leaf(Super okra)than in the okra leaf(T586)and sea-island leaf(H7124).This gene is a class ⅠHOMEODOMAIN-LEUCINE ZIPPER(HD-ZIP Ⅰ)transcription factor likely controlling the deeply lobed leaf shape.in Gossypum named as GhOKRA.We cloned the genomic sequences of OKRA in Chr 1D from different varieties of leaf including Super okra,T586,Hai7124 and TM-1.Multiple sequence alignment revealed that one 8-bp deletion and one 1-bp deletion at positions 742 bp and 861 bp from the ATG start codon were detected,respectively,in TM-1.Sequence analysis showed that the 1-bp deletion present in TM-1 was also found in G.raimondii.It is supposed that the broad leaf is mutated from the wild type okra leaf due to a frameshift mutation.The 133-bp insertion repeated the adjacent region was only found in the promoter region of all super okra and okra varieties compared with sea-island okra and board varieties.It was demonstrated that GhOKRA regulates leaf shape development by virus induced gene silencing(VIGS).The leaves of all Super okra infiltrated with pTRV2-GhOKRA changed from deeply lobed okra to palmate.The rosette leaf blades in transgenic plants over-expressing GhOKRA became narrow,abaxially curled and divided into lobes as compared to the wild type(WT).There was no phenotypic difference between transgenic plants over-expressing GhOKRAM,GrOKRAM and the WT.These results show that premature termination of translation of GhOKRA derived from an 8-bp deletion made this gene non-functional in TM-1.The sequences of OKRA from diploid progenitor D-genome species,and wild races and domesticated allotetraploid cottons in G.hirsutum show that a premature termination mutation occurred before and after the formation of tetraploid cotton,respectively.The 1-bp deletion in OKRA present in wild race species and modern cotton cultivars was found in G.raimondii,indicating that the mutation in tetraploid cottons originated from the D-diploid progenitor species G.raimondii.Another 8-bp deletion mutation occurred after the formation of tetraploid cotton.From the evolution of leaf shape in allotetraploid cottons,we can reasonably deduce that allotetraploid cottons may originate from at least two interspecific hybridizations.One hybridization event occurred between extant progenitors of G.herbaceum or G.arboreum and G.raimondii in which a 1-bp "G" deletion had already occurred before the formation of tetraploid cotton,and produced the present broad leaf tetraploid cottons.Another hybridization event occurred between G.herbaceum or G.arboreum and the wild type or nascent G.raimondii,which had an okra leaf phenotype with complete OKRA,to form the okra leaf tetraploid cottons.After the formation of tetraploid cotton,a further 8-bp deletion occurred in the tetraploid cottons,originating from already broad leaf tetraploid cottons,and resulting in the gene l2,as found in TM-1.To explore how GhOKRA modulates leaf development,we cloned some cotton homologous genes of class-1 KNOTTED 1-like homeobox(KNOX1)gene family,ASYMMETRIC LEAVES1(AS1)and AS2,which were involved in leaf development.Cotton plants silenced by several KANOX1 family genes grew slowly with defected shoot apical meristem(SAM).While the leaf shape did not change.The leaves of Super okra infiltrated with pTRV2-GhASl of pTRV2-GhASl became deeply lobed and split from petiolar sinus.GhAS1 and GhAS2 regulated lobe development in cotton and the complete functional GhOKRA was necessary.The lobes of board leaf cotton infiltrated with pTRV2-GhAS1 or pTRV2-GhAS2 did not change.qPCR showed the expression of GhOKRA in Super okra infiltrated with pTRV2-GhAS1 of pTRV2-GhAS2 did not change,which indicated GhAS1 and GhAS2 did not regulated the transcript level of GhOKRA directly.It was proved that OKRA interacted with AS1 by yeast two-hybrid assay and BiFC.The protein OKRA may control the leaf shape development in cotton through the interaction with AS 1-AS2 complex. |
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