Model

Model Ensemble 1: Linear Regression

Variety name	Total protein content /%	Glutelin content /%	Prolamine content /%	Globulin content /%	Lysine content /%
WT-2x（1）	7.57	2.18	0.44	1.92	0.31
WT-2x（2）	8.3	1.94	0.46	2.09	0.3
WT-2x（3）	8.4	2.15	0.47	2.01	0.32
WT-2x（4）	8.97	2.04	0.4	1.78	0.29
WT-2x（5）	9.13	2.37	0.37	2.19	0.31
WT-2x（6）	9.17	1.85	0.49	2.13	0.3
WT-4x（1）	10.61	3.54	0.49	2.49	0.29
WT-4x（2）	10.55	2.64	0.52	2.51	0.33
WT-4x（3）	11.61	2.77	0.48	2.18	0.36
WT-4x（4）	12.22	2.81	0.45	2.11	0.37
WT-4x（5）	12.45	2.61	0.42	2.68	0.40
WT-4x（6）	14.08	2.21	0.59	2.24	0.35
osglub1-4x（1）	10.65	3.53	0.53	2.4	0.34
osglub1-4x（2）	10.58	2.64	0.57	2.37	0.32
osglub1-4x（3）	11.59	2.76	0.52	2.23	0.33
osglub1-4x（4）	12.23	2.81	0.49	2.13	0.32
osglub1-4x（5）	12.46	2.61	0.46	2.56	0.31
osglub1-4x（6）	14.07	2.22	0.64	2.33	0.34
osglub1/osglb-4x（1）	13.05	2.50	0.62	0.05	0.39
osglub1/osglb-4x（2）	13.23	1.87	0.66	0.04	0.41
osglub1/osglb-4x（3）	14.15	1.96	0.61	0.03	0.40
osglub1/osglb-4x（4）	14.36	1.99	0.57	0.06	0.42
osglub1/osglb-4x（5）	14.89	1.85	0.53	0.07	0.38
osglub1/osglb-4x（6）	15.08	1.56	0.75	0.03	0.40

1.1 Linear Regression of Changes in Total Protein, Gluten, Alcohol-Soluble Protein, Globulin, and Lysine Content Before and After Doubling

1.2 OsGluB1 Linear Regression of Changes in Total Protein, Gluten, Alcohol-Soluble Protein, Globulin, and Lysine Content Before and After Mutation

1.3 OsGluB1/OsGlb Linear Regression of Changes in Total Protein, Gluten, Alcohol-Soluble Protein, Globulin, and Lysine Content Before and After Mutation

1.4 Predictive Comparison of Changes in Total Protein Content Before and After Doubling and Gene Editing

1.5 Predictive Comparison of Changes in Gluten Content Before and After Doubling and Gene Editing

1.6 Predictive Comparison of Changes in Alcohol-Soluble Protein Content Before and After Doubling and Gene Editing

1.7 Predictive Comparison of Changes in Globulin Content Before and After Doubling and Gene Editing

1.8 Predictive Comparison of Changes in Lysine Content Before and After Doubling and Gene Editing

Model accuracy (considering the concentration, significance and goodness of fit of the original data) and improved significance test (α =0.05)
		Variety - Total Protein			Variety - Glutelin			Variety - Prolamine			Variety - Globulin			Variety - Lysine
		First paragraph (polyploidization)	The second segment (osglub1 mutation)	The third segment (both osglub1 and osglb are mutated)	First paragraph (polyploidization)	The second segment (osglub1 mutation)	The third segment (both osglub1 and osglb are mutated)	First paragraph (polyploidization)	The second segment (osglub1 mutation)	The third segment (both osglub1 and osglb are mutated)	First paragraph (polyploidization)	The second segment (osglub1 mutation)	The third segment (both osglub1 and osglb are mutated)	First paragraph (polyploidization)	The second segment (osglub1 mutation)	The third segment (both osglub1 and osglb are mutated)
T-test: paired two sample (content of improvement sequence in this step) mean analysis	Poisson correlation coefficient	0.862274114	0.999916807	0.923103475	0.873683966	-0.199857209	-0.5986107	0.881759617	0.999274682	0.998956606	0.958076122	-0.143809917	-0.947687765	0.920244098	-0.542139318	0.990721002
	P (t<=t) one tailed	0.000690231024756173 (<0.01)	0.335089986659378 (>0.05)	0.000989151779754957 (<0.01)	0.00030753051043595 (<0.01)	0.5 (>0.05)	0.0000051485873642671 (<0.01)	0.0098672689997965 (<0.01)	0.0000282288311043399 (<0.01)	0.0000932871661442039 (<0.01)	0.0157779537244738 (<0.05)	0.344228544888002 (>0.05)	2.63004343672171e-06 (<0.01)	0.00371768700696175 (<0.01)	0.0320250740270976 (<0.05)	0.0035822936958629 (<0.01)
F-test two sample ANOVA (to prepare for the next t-test)	P (f<=f) one tailed	0.061840102311957 (>0.05)	0.490229545293888 (>0.05)	0.176102716725459 (>0.05)	0.042323872868052 (<0.05)	0.488022077700087 (>0.05)	0.239757368280236 (>0.05)	0.287220640942428 (>0.05)	0.440003756859274 (>0.05)	0.347834034250312 (>0.05)	0.19530882719514 (>0.05)	0.19130366703838 (>0.05)	0.0000858277908973245 (<0.01)	0.00718242019246973 (<0.01)	0.0194917704950284 (<0.05)	0.0260907002285288 (<0.05)
T-test: two sample equal / heteroscedasticity hypothesis (to evaluate the significance of the difference in the content of improvement sequence in this step)	P (t<=t) two tailed	0.000827401482597938 (<0.01)	0.989738281660783 (>0.05)	0.00604198967350708 (<0.01)	0.0101455895746064 (<0.05)	0.994810846434833 (>0.05)	0.00583507750656658 (<0.01)	0.010133803616988 (<0.05)	0.249195635126652 (>0.05)	0.00543013350300424 (<0.01)	0.0101401106876729 (<0.05)	0.77837278673196 (>0.05)	2.46115913592971e-07 (<0.01)	0.0296988118180533 (<0.05)	0.04636248597522 (<0.05)	0.0091720143500425 (<0.05)
Regression statistics - goodness of fit analysis (to evaluate the causal relationship between the improvement and content change in this step)	Multiple R	0.961984213	0.99991555	0.931414707	0.935282517	0.999948408	0.935282517	0.973746336	0.918466367	0.960978441	0.670697327	0.910572177	0.996507557	0.667736145	0.940790155	0.940790155
	R Square	0.925413626	0.999831107	0.867533356	0.918947087	0.999896818	0.918947087	0.924435591	0.9751141	0.937183703	0.649811202	0.900829142	0.993027312	0.64587156	0.916638367	0.916638367
	Adjusted R Square	0.906767032	0.999788883	0.834416695	0.901316141	0.999871023	0.901316141	0.94687915	0.909262551	0.930902073	0.629792322	0.909087944	0.992330043	0.639045872	0.904202041	0.904202041
General comments		Extremely excellent	Excellent accuracy but poor significance	Extremely excellent	excellent	Excellent accuracy but poor significance	Extremely excellent	excellent	Excellent accuracy but poor significance	Extremely excellent	excellent	Excellent accuracy but poor significance	Extremely excellent	excellent	Excellent accuracy but poor significance	Extremely excellent

Model Ensemble 2 Prediction of Protein Primary Structure (Highlighted in Yellow are Target Locus Genes)

2.1 Prediction of Primary Structure for Wild-Type Glutenin B1

Nucleotide Sequence of the Glutenin B1-Encoding Gene OsGluB1

1 aactagcttg gtctgtcttt gaactcacat caattagctt aagtttccat aagcaagtac
61 aaatagctat ggcgagttcc gttttctctc ggttttctat atacttttgt gttcttctat
121 tatgccatgg ttctatggcc cagctattta atcccagcac aaacccatgg catagtcctc
181 ggcaaggaag ttttagggag tgtagatttg atagactaca agcatttgaa ccacttcgga
241 aagtgaggtc agaagctggg gtgactgagt acttcgatga gaagaatgaa ttattccagt
301 gcacgggtac ttttgtgatc cgacgtgtca ttcagcctca aggccttttg gtacctcgat
361 acacaaatat tcctggcgtg gtctacatca tccaaggttt gtatcctaat aaatataatc
421 gagaactcta tcgatgcaac tagtagttat gatctccatg gtcaaattaa tgttgtatgt
481 accttatgtg aacattataa tttttaggga gaggttctat gggtttaacc ttccccggtt
541 gccctgcgac ttaccagcaa caattccaac aattttcatc tcaaggccaa agtcagagcc
601 aaaagtttag agatgagcac caaaagattc atcaatttag gcaaggagac attgttgcac
661 tcccagctgg tgttgcacat tggttctaca atgatggtga tgcacctatt gttgccgtat
721 atgtttatga cgtaaacaac aacgccaatc agcttgaacc taggcaaaag gtaatttttt
781 gaacaaatat ttccatcaaa gttgtaattt tctatgtttt tcatatgttc ttacaatgca
841 attcatagcg taaaacatta aatttttcat tgataaaaat ttcaggagtt cctattagcc
901 ggcaacaaca atcgggctca acaacaacaa gtatatggta gctcaattga gcaacactct
961 gggcaaaaca tattcagcgg atttggtgtt gagatgctaa gtgaggcttt aggcatcaac
1021 gcagtagcag caaagaggct acagagccaa aatgatcaaa gaggagagat catacatgtg
1081 aagaatggcc ttcaattgtt gaaaccgact ttgacacaac agcaagaaca agcacaagca
1141 caagatcaat atcaacaagt tcaatacagt gaacgacagc aaacatcttc tcgatggaac
1201 ggattggagg agaacttttg cacgatcaag gtgagagtaa acattgaaaa tcctagtcgt
1261 gctgattcat acaacccacg tgccggaagg ataacaagtg tcaatagtca gaagttcccc
1321 atccttaacc tcatccaaat gagcgctacc agagtaaacc tataccaggt atttattata
1381 gcaccgtttt gttctcttca ttttttttgg aagatctttg atttgttgct tatataacat
1441 tattctctac tgatttctac agaatgctat tctctcgccg ttctggaacg tcaatgctca
1501 tagtttggtc tatatgattc aagggcgatc tcgagttcaa gtcgttagta actttggaaa
1561 gactgtgttt gatggtgtcc ttcgcccagg acaattattg atcattccgc aacattatgc
1621 tgtcttgaag aaagcagagc gtgaaggatg ccaatatatc gcaatcaaga caaacgctaa
1681 cgccttcgtc agccaccttg cagggaaaaa ctcagtattc cgtgccttgc cagttgatgt
1741 agtcgctaat gcgtatcgca tctcaaggga gcaagcccga agcctcaaga acaacagggg
1801 agaagagcac ggtgccttca ctcctagatt tcaacaacaa tactacccag gattatcgaa
1861 tgagtccgaa agcgagacct cagagtaatg taattgagaa ctagtatcgg cgtagagtaa
1921 aataaaacac cacaagtatg acacttggtg gtgattctgt tcgatatcag tactaaataa
1981 aggttacaaa cttcttaatt ttccta

Amino Acid Sequence of Wild-Type Glutenin B1

              MASSVFSRFSIYFCVLLLCHGSMAQLFNPSTNPWHSPRQGSFRECRFDRLQAFEPLRKVRSEAGVTEYFDEKNELFQCTGTFVIRRVIQPQGLLVPRYTNIPGVVYIIQGRGSMGLTFPGCPATYQQQFQQFSSQGQSQSQKFRDEHQKIHQFRQGDIVALPAGVAHWFYNDGDAPIVAVYVYDVNNNANQLEPRQKEFLLAGNNNRAQQQQVYGSSIEQHSGQNIFSGFGVEMLSEALGINAVAAKRLQSQNDQRGEIIHVKNGLQLLKPTLTQQQEQAQAQDQYQQVQYSERQQTSSRWNGLEENFCTIKVRVNIENPSRADSYNPRAGRITSVNSQKFPILNLIQMSATRVNLYQNAILSPFWNVNAHSLVYMIQGRSRVQVVSNFGKTVFDGVLRPGQLLIIPQHYAVLKKAEREGCQYIAIKTNANAFVSHLAGKNSVFRALPVDVVANAYRISREQARSLKNNRGEEHGAFTPRFQQQYYPGLSNESESETSE
            

2.2 Prediction of Primary Structure for Mutant Glutenin B1

OsGluB1 Nucleotide Sequence of Gene Mutation
（Case 1 - Mutant 1: Deletion of One G, Frameshift Mutation）

1 aactagcttg gtctgtcttt gaactcacat caattagctt aagtttccat aagcaagtac
61 aaatagctat ggcgagttcc gttttctctc ggttttctat atacttttgt gttcttctat
121 tatgccatgg ttctatggcc cagctattta atcccagcac aaacccatgg catagtcctc
181 ggcaaggaag ttttagggag tgtagatttg atagactaca agcatttgaa ccacttcgga
241 aagtgaggtc agaagctggg gtgactgagt acttcgatga gaagaatgaa ttattccagt
301 gcacgggtac ttttgtgatc cgacgtgtca ttcagcctca aggccttttg gtacctcgat
361 acacaaatat tcctggcgtg gtctacatca tccaaggttt gtatcctaat aaatataatc
421 gagaactcta tcgatgcaac tagtagttat gatctccatg gtcaaattaa tgttgtatgt
481 accttatgtg aacattataa tttttaggga gaggttctat gggtttaacc ttccccgtt
541 gccctgcgac ttaccagcaa caattccaac aattttcatc tcaaggccaa agtcagagcc
601 aaaagtttag agatgagcac caaaagattc atcaatttag gcaaggagac attgttgcac
661 tcccagctgg tgttgcacat tggttctaca atgatggtga tgcacctatt gttgccgtat
721 atgtttatga cgtaaacaac aacgccaatc agcttgaacc taggcaaaag gtaatttttt
781 gaacaaatat ttccatcaaa gttgtaattt tctatgtttt tcatatgttc ttacaatgca
841 attcatagcg taaaacatta aatttttcat tgataaaaat ttcaggagtt cctattagcc
901 ggcaacaaca atcgggctca acaacaacaa gtatatggta gctcaattga gcaacactct
961 gggcaaaaca tattcagcgg atttggtgtt gagatgctaa gtgaggcttt aggcatcaac
1021 gcagtagcag caaagaggct acagagccaa aatgatcaaa gaggagagat catacatgtg
1081 aagaatggcc ttcaattgtt gaaaccgact ttgacacaac agcaagaaca agcacaagca
1141 caagatcaat atcaacaagt tcaatacagt gaacgacagc aaacatcttc tcgatggaac
1201 ggattggagg agaacttttg cacgatcaag gtgagagtaa acattgaaaa tcctagtcgt
1261 gctgattcat acaacccacg tgccggaagg ataacaagtg tcaatagtca gaagttcccc
1321 atccttaacc tcatccaaat gagcgctacc agagtaaacc tataccaggt atttattata
1381 gcaccgtttt gttctcttca ttttttttgg aagatctttg atttgttgct tatataacat
1441 tattctctac tgatttctac agaatgctat tctctcgccg ttctggaacg tcaatgctca
1501 tagtttggtc tatatgattc aagggcgatc tcgagttcaa gtcgttagta actttggaaa
1561 gactgtgttt gatggtgtcc ttcgcccagg acaattattg atcattccgc aacattatgc
1621 tgtcttgaag aaagcagagc gtgaaggatg ccaatatatc gcaatcaaga caaacgctaa
1681 cgccttcgtc agccaccttg cagggaaaaa ctcagtattc cgtgccttgc cagttgatgt
1741 agtcgctaat gcgtatcgca tctcaaggga gcaagcccga agcctcaaga acaacagggg
1801 agaagagcac ggtgccttca ctcctagatt tcaacaacaa tactacccag gattatcgaa
1861 tgagtccgaa agcgagacct cagagtaatg taattgagaa ctagtatcgg cgtagagtaa
1921 aataaaacac cacaagtatg acacttggtg gtgattctgt tcgatatcag tactaaataa
1981 aggttacaaa cttcttaatt ttccta

（Case 2 - Mutant 2: Deletion of One T, Frameshift Mutation）

1 aactagcttg gtctgtcttt gaactcacat caattagctt aagtttccat aagcaagtac
61 aaatagctat ggcgagttcc gttttctctc ggttttctat atacttttgt gttcttctat
121 tatgccatgg ttctatggcc cagctattta atcccagcac aaacccatgg catagtcctc
181 ggcaaggaag ttttagggag tgtagatttg atagactaca agcatttgaa ccacttcgga
241 aagtgaggtc agaagctggg gtgactgagt acttcgatga gaagaatgaa ttattccagt
301 gcacgggtac ttttgtgatc cgacgtgtca ttcagcctca aggccttttg gtacctcgat
361 acacaaatat tcctggcgtg gtctacatca tccaaggttt gtatcctaat aaatataatc
421 gagaactcta tcgatgcaac tagtagttat gatctccatg gtcaaattaa tgttgtatgt
481 accttatgtg aacattataa tttttaggga gaggttctat gggtttaacc ttccccggt
541 gccctgcgac ttaccagcaa caattccaac aattttcatc tcaaggccaa agtcagagcc
601 aaaagtttag agatgagcac caaaagattc atcaatttag gcaaggagac attgttgcac
661 tcccagctgg tgttgcacat tggttctaca atgatggtga tgcacctatt gttgccgtat
721 atgtttatga cgtaaacaac aacgccaatc agcttgaacc taggcaaaag gtaatttttt
781 gaacaaatat ttccatcaaa gttgtaattt tctatgtttt tcatatgttc ttacaatgca
841 attcatagcg taaaacatta aatttttcat tgataaaaat ttcaggagtt cctattagcc
901 ggcaacaaca atcgggctca acaacaacaa gtatatggta gctcaattga gcaacactct
961 gggcaaaaca tattcagcgg atttggtgtt gagatgctaa gtgaggcttt aggcatcaac
1021 gcagtagcag caaagaggct acagagccaa aatgatcaaa gaggagagat catacatgtg
1081 aagaatggcc ttcaattgtt gaaaccgact ttgacacaac agcaagaaca agcacaagca
1141 caagatcaat atcaacaagt tcaatacagt gaacgacagc aaacatcttc tcgatggaac
1201 ggattggagg agaacttttg cacgatcaag gtgagagtaa acattgaaaa tcctagtcgt
1261 gctgattcat acaacccacg tgccggaagg ataacaagtg tcaatagtca gaagttcccc
1321 atccttaacc tcatccaaat gagcgctacc agagtaaacc tataccaggt atttattata
1381 gcaccgtttt gttctcttca ttttttttgg aagatctttg atttgttgct tatataacat
1441 tattctctac tgatttctac agaatgctat tctctcgccg ttctggaacg tcaatgctca
1501 tagtttggtc tatatgattc aagggcgatc tcgagttcaa gtcgttagta actttggaaa
1561 gactgtgttt gatggtgtcc ttcgcccagg acaattattg atcattccgc aacattatgc
1621 tgtcttgaag aaagcagagc gtgaaggatg ccaatatatc gcaatcaaga caaacgctaa
1681 cgccttcgtc agccaccttg cagggaaaaa ctcagtattc cgtgccttgc cagttgatgt
1741 agtcgctaat gcgtatcgca tctcaaggga gcaagcccga agcctcaaga acaacagggg
1801 agaagagcac ggtgccttca ctcctagatt tcaacaacaa tactacccag gattatcgaa
1861 tgagtccgaa agcgagacct cagagtaatg taattgagaa ctagtatcgg cgtagagtaa
1921 aataaaacac cacaagtatg acacttggtg gtgattctgt tcgatatcag tactaaataa
1981 aggttacaaa cttcttaatt ttccta

Amino Acid Sequence of Mutant Glutenin B1
（Frameshift Mutation: Case 1 - Mutant 1）

              MASSVFSRFSIYFCVLLLCHGSMAQLFNPSTNPWHSPRQGSFRECRFDRLQAFEPLRKVR
              SEAGVTEYFDEKNELFQCTGTFVIRRVIQPQGLLVPRYTNIPGVVYIIQGLYPNKYNREL
              YRCNLSPWSNCCMYLMTLFLGRGSMGLTFPVALRLTSNNSNNFHLKAKVRAKSLEMSTKR
              FINLGKETLLHSQLVLHIGSTMMMHLLLPYMFMTTTTPTSLNLGKRFFEQIFPSKLFSW
              FFICSYNAIHSVKHIFHKFQEFLLAGNNNRAQQQQVYGSSIEQHSGQNIFSGFGVEMLSE
              ALGINAVAARRLQSQNDQRGEIIHVKNGLQLLKPTLTQQQEQAQAQDQYQQVQYSERQQT
              SSRWNGLEENFCTIKVRVNIENPSRADSYNPRAGRITSVNSQKFPILNLIQMSATRVNLY
              QVFIIAPFCSLHFFWKIFDLLLIHYSLLISTECYSLAVLERQCSFGLYDS RAISSSSRLW
              KDCVWCPSPRTIIDHSATLCCLEESRARMPIYRNQDKRRLRQPPCREKLSIPCLASCSRC
              VSHLKGASPKPQEQQGRRARCLHSISTTILPRIIEVRKRDLRV

            

（Frameshift Mutation: Case 2 - Mutant 2）

              MASSVFSRFSIYFCVLLLCHGSMAQLFNPSTNPWHSPRQGSFRECRFDRLQAFEPLRKVR
              SEAGVTEYFDEKNELFQCTGTFVIRRVIQPQGLLVPRYTNIPGVVYIIQGLYPNKYNREL
              YRCNLSPWSNCCMYLMTLFLGRGSMGLTFPGALRLTSNNSNNFHLKAKVRAKSLEMSTKR
              FINLGKETLLHSQLVLHIGSTMMMHLLLPYMFMTTTTPTSLNLGKRFFEQIFPSKLFSW
              FFICSYNAIHSVKHIFHKFQEFLLAGNNNRAQQQQVYGSSIEQHSGQNIFSGFGVEMLSE
              ALGINAVAARRLQSQNDQRGEIIHVKNGLQLLKPTLTQQQEQAQAQDQYQQVQYSERQQT
              SSRWNGLEENFCTIKVRVNIENPSRADSYNPRAGRITSVNSQKFPILNLIQMSATRVNLY
              QVFIIAPFCSLHFFWKIFDLLLIHYSLLISTECYSLAVLERQCSFGLYDSRAISSSSRLW
              KDCVWCPSPRTIIDHSATLCCLEESRARMPIYRNQDKRRLRQPPCREKLSIPCLASCSRC
              VSHLKGASPKPQEQQGRRARCLHSISTTILPRIIEVRKRDLRV

            

2.3 Prediction of Primary Structure for Wild-Type Globulin

Nucleotide Sequence of the Globulin-Encoding Gene OsGlb

1 cgatcgccat catctcatca tcagttcatc accaacaaac aaaagaggaa aaaaaacata
61 tacacttcta gtgattgtct gattgatcat caatggctag caaggtcgtc ttcttcgcgg
121 cggcgctcat ggcggccatg gtggccatct ccggcgcgca gctgagcgag tcggagatga
181 ggttcaggga caggcagtgc cagcgggagg tgcaggacag cccgctggac gcgtgccggc
241 aggtgctcga ccggcagctc accggccggg agaggttcca gccgatgttc cgccgcccgg
301 gcgcgctcgg cctgcggatg cagtgctgcc agcagctgca ggacgtgagc cgcgagtgcc
361 gctgcgccgc catccgccgg atggtgagga gctacgagga gagcatgccg atgcccctgg
421 agcaaggctg gtcgtcgtcg tcgtcggagt actacggcgg cgaggggtcg tcgtcggagc
481 aggggtacta cggcgagggg tcgtcggagg agggctacta cggcgagcag cagcagcagc
541 cggggatgac ccgcgtgagg ctgaccaggg cgaggcagta cgcggcgcag ctgccgtcga
601 tgtgccgggt tgagccccag cagtgcagca tcttcgccgc cggccagtac taggcttagc
661 tggcttgcct catagctagt aggatcacac cgttgctgtg ttgcatgcaa cgagatagtg
721 atcaaaacaa ataatgaata atgaaatgtt ttgaaaacaa aatagctggt gataaagtac
781 tatatatgat tttgtgcgtt ctg

              MASKVVFFAAALMAAMVAISGAQLSESEMRFRDRQCQREVQDSPLDACRQVLDRQLTGRERFQPMFRRPGALGLRMQCCQQLQDVSRECRCAAIRRMVRSYEESMPMPLEQGWSSSSSEYYGGEGSSSEQGYYGEGSSEEGYYGEQQQQPGMTRVRLTRARQYAAQLPSMCRVEPQQCSIFAAGQY

            

2.4 Prediction of Primary Structure for Mutant Globulin

OsGlb Nucleotide Sequence of Gene Mutation

1 cgatcgccat catctcatca tcagttcatc accaacaaac aaaagaggaa aaaaaacata
61 tacacttcta gtgattgtct gattgatcat caatggctag caaggtcgtc ttcttcgcgg
121 cggcgctcat ggcggccatg gtggccatct ccggcgcgca gctgagcgag tcggagatga
181 ggttcaggga caggcagtgc cagcgggagg tgcaggacag cccgctggac gcgtgccggc
241 aggtgctcga ccggcagctc accggccggg agaggttcca gccgatgttc cgccgcccgg
301 gcgcgctcgg cctgcggatg cagtgctgcc agcagctgca ggacgtgagc cgcgagtgcc
361 gctgcgccgc catccgccgg atggtgagga gctacgagga gagcatgccg atgcccctgg
421 agcaaggctg gtcgtcgtcg tcgtcggagt actaacggcgg cgaggggtcg tcgtcggagc
481 aggggtacta cggcgagggg tcgtcggagg agggctacta cggcgagcag cagcagcagc
541 cggggatgac ccgcgtgagg ctgaccaggg cgaggcagta cgcggcgcag ctgccgtcga
601 tgtgccgggt tgagccccag cagtgcagca tcttcgccgc cggccagtac taggcttagc
661 tggcttgcct catagctagt aggatcacac cgttgctgtg ttgcatgcaa cgagatagtg
721 atcaaaacaa ataatgaata atgaaatgtt ttgaaaacaa aatagctggt gataaagtac
781 tatatatgat tttgtgcgtt ctg

Amino Acid Sequence of Mutant Globulin

MASKVVFFAA ALMAAMVAIS GAQLSESEMR FRDRQCQREV QDSPLDACRQ VLDRQLTGRE RFQPMFRRPG ALGLRMQCCQ QLQDVSRECR CAAIRRMVRS YEESMPMPLE QGWSSSSSEY RRRGVVVGAG VLRRGVVGGG LLRRAAAAAG DDPREADQGE AVRGAAAVDV PGAPAVQHLR

Model Summary: Prediction Results of Primary Structures for Wild-Type and Mutant Glutenin B1, and Globulin

	GlutelinB1					Globulin
	WT	WT	Mutant-1(50%)	Mutant-2(50%)	Mutant-2(50%)	WT	Mutant
Number of amino acids	499	499	583	583	583	186	185
Molecular weight	56550.58	56550.58	67178.78	67136.70	67136.70	21054.74	20346.4
PI	9.26	9.26	9.66	9.66	9.66	7.48	10.22
Total number of negatively charged residues	39	39	38	38	38	21	20
Total number of positively charged residues	50	50	70	70	70	22	29
Instability index	52.11	52.11	58.90	58.42	58.42	69.24	72.62
Aliphatic index	76.19	76.19	85.95	85.45	85.45	52.53	75.46
Grand average of hydropathicity	-0.495	-0.495	-0.226	-0.234	-0.234	-0.629	-0.317
Amino acids composition
	Glutelin-WT	Glutelin-Mutant1	Glutelin-Mutant2	Globulin-WT	Globulin-Mutant

Rice	GlutelinB1 - Mutation	Globulin - Mutation
Biological Value(BV)	↑	↓
taste	Unable to determine	Unable to determine
digestibility	Unable to determine	Unable to determine
allergenicity	Unable to determine	Unable to determine

Model Ensemble 3

3.1 Tertiary Structure of Wild-Type Glutenin B1

3.2 Prediction of Tertiary Structure for Mutant Glutenin B1 and Confidence Level

Case 1-Glutelin M1

Case 2-Glutelin M2

3.3 Prediction of Tertiary Structure for Wild-Type Globulin and Confidence Level

(Obtained Directly from Protein Database)

3.4 Prediction of Tertiary Structure for Mutant Globulin and Confidence Level

Model Ensemble 4: Molecular Docking

4.1 Molecular Docking of Wild-Type Glutenin B1 with Human Immunoglobulin E Heavy Chain Constant Region

Binding Sites

Glutelin	IgE
173Asp	398Gln
285Tyr	231Arg
323Asp	272Gln
377Gln	388Arg
421Gln	370His
431Ala	273Arg
439Lys	215Gly, 242Asp, 241Val
440Asn	242Asp
471Glu	217Ser

4.2 Molecular Docking of Mutant Glutenin B1 with Human Immunoglobulin E Heavy Chain Constant Region

Binding Sites
Case 1

Residue in Glutelin-M1	Interacting Residue in IgE
37Arg	41Trp, 44Thr
97Tyr	94Asp
159Ser	5Ser
160Asn	4Gln
161Asn	202Glu
165Lys	191Thr
172Ser	187Asp
195Leu	188Arg
196His	188Arg

case 2

Glutelin-M2	IgE
37Arg	42Trp, 44Thr
97Tyr	94Asp
159Ser	5Ser
160Asn	4Gln, 7Ser
161Asn	202Glu
165Lys	191Thr
172Ser	187Asp
195Leu	188Arg
196His	188Arg

4.3 Molecular Docking of Wild-Type Globulin with Human Immunoglobulin E Heavy Chain Constant Region